Human virus causing severe acute respiratory syndrome (SARS) and uses thereof

ABSTRACT

The present invention relates to an isolated novel virus causing Severe Acute Respiratory Syndrome (SARS) in humans (“hSARS virus”). The hSARS virus is identified to be morphologically and phylogenetically similar to known member of Coronaviridae. The present invention provides the complete genomic sequence of the hSARS virus. Furthermore, the invention provides the nucleic acids and peptides encoded by and/or derived from the hSARS virus and their use in diagnostic methods and therapeutic methods, including vaccines. In addition, the invention provides chimeric or recombinant viruses encoded by said nucleotide sequences and antibodies immunospecific to the polypeptides encoded by the nucleotide sequences.

This application is a division of U.S. patent application Ser. No.10/808,121, filed Mar. 24, 2004; which claims priority benefit to U.S.provisional application No. 60/457,031, filed Mar. 24, 2003; U.S.provisional application No. 60/457,730, filed Mar. 26, 2003; U.S.provisional application No. 60/459,931, filed Apr. 2, 2003; U.S.provisional application No. 60/460,357, filed Apr. 3, 2003; U.S.provisional application No. 60/461,265, filed Apr. 8, 2003; U.S.provisional application No. 60/462,805, filed Apr. 14, 2003; and U.S.provisional application No. 60/464,886 filed Apr. 23, 2003, each ofwhich is incorporated herein by reference in its entirety.

The instant application contains a lengthy Sequence Listing which isbeing concurrently submitted via triplicate CD-R in lieu of a printedpaper copy, and is hereby incorporated by reference in its entirety.Said CD-R, recorded on Jun. 5, 2007, are labeled “CRF”, “Copy 1” and“Copy 2”, respectively, and each contains only one identical 1.58 MBfile (V96611027.APP).

1. INTRODUCTION

The present invention relates to an isolated novel virus causing SevereAcute Respiratory Syndrome (SARS) in humans (“hSARS virus”). The hSARSvirus is identified to be morphologically and phylogenetically similarto known members of Coronaviridae. The present invention relates to anucleotide sequence comprising the complete genomic sequence of thehSARS virus. The invention further relates to nucleotide sequencescomprising a portion of the genomic sequence of the hSARS virus. Theinvention also relates to the deduced amino acid sequences of thecomplete genome of the hSARS virus. The invention further relates to thenucleic acids and peptides encoded by and/or derived from thesesequences and their use in diagnostic methods and therapeutic methods,such as for immunogens. The invention further encompasses chimeric orrecombinant viruses encoded by said nucleotide sequences and antibodiesdirected against polypeptides encoded by the nucleotide sequence.Furthermore, the invention relates to vaccine preparations comprisingthe hSARS virus, including recombinant and chimeric forms of said virusas well as protein extracts and subunits of said virus.

2. BACKGROUND OF THE INVENTION

Recently, there has been an outbreak of atypical pneumonia in Guangdongprovince in mainland China. Between November 2002 and March 2003, therewere 792 reported cases with 31 fatalities (WHO. Severe AcuteRespiratory Syndrome (SARS) Weekly Epidemiol Rec. 2003; 78: 86). Inresponse to this crisis, the Hospital Authority in Hong Kong hasincreased the surveillance on patients with severe atypical pneumonia.In the course of this investigation, a number of clusters of health careworkers with the disease were identified. In addition, there wereclusters of pneumonia incidents among persons in close contact withthose infected. The disease was unusual in its severity and itsprogression in spite of the antibiotic treatment typical for thebacterial pathogens that are known to be commonly associated withatypical pneumonia. The present inventors were one of the groupsinvolved in the investigation of these patients. All tests foridentifying commonly recognized viruses and bacteria were negative inthese patients. The disease was given the acronym Severe AcuteRespiratory Syndrome (“SARS”). The etiologic agent responsible for thisdisease was not known until the isolation of hSARS virus from the SARSpatients by the present inventors as disclosed herein. Namely, thepresent invention discloses a novel human virus that has been isolatedand identified from the patients suffering from SARS. The invention isuseful in both clinical and scientific research applications.

3. SUMMARY OF INVENTION

The present invention is based upon the inventor's isolation andidentification of a novel virus causing Severe Acute RespiratorySyndrome in humans (“hSARS virus”). The virus was isolated from thepatients suffering from SARS in the recent outbreak of severe atypicalpneumonia in China. The isolated virus is an enveloped, single-strandedRNA virus of positive polarity which belongs to the order, Nidovirales,of the family, Coronaviridae. Accordingly, the invention relates to theisolated hSARS virus that morphologically and phylogenetically relatesto known members of Coronaviridae. In a specific embodiment, theisolated hSARS virus is that which was deposited with China Center forType Culture Collection (CCTCC) on Apr. 2, 2003 and accorded anaccession number, CCTCC-V200303, as described in Section 7, infra. Inanother specific embodiment, the invention provides complete genomicsequence of the hSARS virus. In a preferred embodiment, the viruscomprises a nucleotide sequence of SEQ ID NO:15. In another specificembodiment, the invention provides nucleic acids isolated from thevirus. The virus preferably comprises a nucleotide sequence of SEQ IDNO:1, 11 and/or 13 in its genome. In a specific embodiment, the presentinvention provides isolated nucleic acid molecules comprising or,alternatively, consisting of the nucleotide sequence of SEQ ID NO:1, acomplement thereof or a portion thereof, preferably at least 5, 10, 15,20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550,600, or more contiguous nucleotides of the nucleotide sequence of SEQ IDNO:1, or a complement thereof. In another specific embodiment, thepresent invention provides isolated nucleic acid molecules comprisingor, alternatively, consisting of the nucleotide sequence of SEQ IDNO:11, a complement thereof or a portion thereof, preferably at least 5,10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150,1,200, or more contiguous nucleotides of the nucleotide sequence of SEQID NO:11, or a complement thereof. In yet another specific embodiment,the present invention provides isolated nucleic acid moleculescomprising or, alternatively, consisting of the nucleotide sequence ofSEQ ID NO:13, a complement thereof or a portion thereof, preferably atleast 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400,450, 500, 550, 600, 650, 700, or more contiguous nucleotides of thenucleotide sequence of SEQ ID NO:13, or a complement thereof. In anotherspecific embodiment, the present invention provides isolated nucleicacid molecules comprising or, alternatively, consisting of thenucleotide sequence of SEQ ID NO:15, a complement thereof or a portionthereof, preferably at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100,150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 2,000, 3,000, 4,000, 5,000,6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000,15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000,24,000, 25,000, 26,000, 27,000, 28,000, 29,000 or more contiguousnucleotides of the nucleotide sequence of SEQ ID NO:15, or a complementthereof. Furthermore, in another specific embodiment, the inventionprovides isolated nucleic acid molecules which hybridize under stringentconditions, as defined herein, to a nucleic acid molecule having thesequence of SEQ ID NO:1, 11, 13, 15, 16, 240, 737, 1108, 1590 or 1965 ora complement thereof. In one embodiment, the invention provides anisolated nucleic acid molecule which is antisense to the coding strandof a nucleic acid of the invention. In another specific embodiment, theinvention provides isolated polypeptides or proteins that are encoded bya nucleic acid molecule comprising or, alternatively consisting of anucleotide sequence that is at least 5, 10, 15, 20, 25, 30, 35, 40, 45,100, 150, 200, 300, 350, 400, 450, 500, 550, 600, or more contiguousnucleotides of the nucleotide sequence of SEQ ID NO:1, or a complementthereof. In yet another specific embodiment, the invention providesisolated polypeptides or proteins that are encoded by a nucleic acidmolecule comprising or, alternatively consisting of a nucleotidesequence that is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150,200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1,000, 1,050, 1,100, 1,150, 1,200 or more contiguous nucleotides ofthe nucleotide sequence of SEQ ID NO:11, or a complement thereof. In yetanother specific embodiment, the invention provides isolatedpolypeptides or proteins that are encoded by a nucleic acid moleculecomprising or, alternatively consisting of a nucleotide sequence that isat least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350,400, 450, 500, 550, 600, 650, 700, or more contiguous nucleotides of thenucleotide sequence of SEQ ID NO:13, or a complement thereof. In yetanother specific embodiment, the invention provides isolatedpolypeptides or proteins that are encoded by a nucleic acid moleculecomprising or, alternatively consisting of a nucleotide sequence that isat least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,1,050, 1,100, 1,150, 1,200, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000,8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000,17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000,26,000, 27,000, 28,000, 29,000 or more contiguous nucleotides of thenucleotide sequence of SEQ ID NO:15, or a complement thereof. Theinvention further provides proteins or polypeptides that are isolatedfrom the hSARS virus, including viral proteins isolated from cellsinfected with the virus but not present in comparable uninfected cells.The invention further provides proteins or polypeptides of SEQ ID NOS:2,12 and 14 and those shown in FIGS. 11 (SEQ ID NOS:17-239, 241-736 and738-1107) and 12 (1109-1589, 1591-1964, 1966-2470). The polypeptides orthe proteins of the present invention preferably have a biologicalactivity of the protein (including antigenicity and/or immunogenicity)encoded by the sequence of SEQ ID NO:1, 11, 13, 16, 240, 737, 1108, 1590or 1965. In other embodiments, the polypeptides or the proteins of thepresent invention have a biological activity of the protein (includingantigenicity and/or immunogenicity) encoded by a nucleotide sequencethat is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,1,050, 1,100, 1,150, 1,200, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000,8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000,17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000,26,000, 27,000, 28,000, 29,000 or more contiguous nucleotides of thenucleotide sequence of SEQ ID NO:15, or a complement thereof. In otherembodiments, the polypeptides or the proteins of the present inventionhave a biological activity of the protein (including antigenicity and/orimmunogenicity) of FIGS. 11 (SEQ ID NOS:17-239, 241-736 and 738-1107)and 12 (SEQ ID NOS:1109-1589, 1591-1964 and 1966-2470).

In one aspect, the invention provides a method for propagating the hSARSvirus in host cells comprising infecting the host cells with theisolated hSARS virus, culturing the host cells to allow the virus tomultiply, and harvesting the resulting virions. Also provide by thepresent invention are host cells that are infected with the hSARS virus.In another aspect, the invention relates to the use of the isolatedhSARS virus for diagnostic and therapeutic methods. In a specificembodiment, the invention provides a method of detecting in a biologicalsample an antibody immunospecific for the hSARS virus using the isolatedhSARS virus or any proteins or polypeptides thereof. In another specificembodiment, the invention provides a method of screening for an antibodywhich immunospecifically binds and neutralizes hSARS. Such an antibodyis useful for a passive immunization or immunotherapy of a subjectinfected with hSARS.

The invention further relates to the use of the sequence information ofthe isolated virus for diagnostic and therapeutic methods. In a specificembodiment, the invention provides nucleic acid molecules which aresuitable for use as primers consisting of or comprising the nucleotidesequence of SEQ ID NO:1, 11, 13, or 15, a complement thereof, or atleast a portion of the nucleotide sequence thereof. In another specificembodiment, the invention provides nucleic acid molecules which aresuitable for hybridization to hSARS nucleic acid, including, but notlimited to, as PCR primers, Reverse Transcriptase primers, probes forSouthern analysis or other nucleic acid hybridization analysis for thedetection of hSARS nucleic acids, e.g., consisting of or comprising thenucleotide sequence of SEQ ID NO:1, 11, 13, or 15, a complement thereof,or a portion thereof. The invention further encompasses chimeric orrecombinant viruses encoded in whole or in part by said nucleotidesequences.

The invention further provides antibodies that specifically bind apolypeptide of the invention encoded by the nucleotide sequence of SEQID NO:1, 11, 13, 16, 240, 737, 1108, 1590 or 1965, or a fragmentthereof, or encoded by a nucleic acid comprising a nucleotide sequencethat hybridizes under stringent conditions to the nucleotide sequence ofSEQ ID NO:1, 11, or 13, and/or any hSARS epitope, having one or morebiological activities of a polypeptide of the invention. The inventionfurther provides antibodies that specifically bind polypeptides of theinvention encoded by the nucleotide sequence of SEQ ID NO:15 or acomplement thereof, or a fragment thereof. These polypeptides includethose shown in FIGS. 11 (SEQ ID NOS:17-239, 241-736 and 738-1107) and 12(SEQ ID NOS:1109-1589, 1591-1964 and 1966-2470). The invention furtherprovides antibodies that specifically bind polypeptides of the inventionencoded by a nucleic acid comprising a nucleotide sequence thathybridizes under stringent conditions to the nucleotide sequence of SEQID NO:15, and/or any hSARS epitope, having one or more biologicalactivities of a polypeptide of the invention. Such antibodies include,but are not limited to polyclonal, monoclonal, bi-specific,multi-specific, human, humanized, chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, disulfide-linked Fvs,intrabodies and fragments containing either a VL or VH domain or even acomplementary determining region (CDR) that specifically binds to apolypeptide of the invention.

In one embodiment, the invention provides methods for detecting thepresence, activity or expression of the hSARS virus of the invention ina biological material, such as cells, blood, saliva, urine, and soforth. The increased or decreased activity or expression of the hSARSvirus in a sample relative to a control sample can be determined bycontacting the biological material with an agent which can detectdirectly or indirectly the presence, activity or expression of the hSARSvirus. In a specific embodiment, the detecting agents are the antibodiesor nucleic acid molecules of the present invention. Antibodies of theinvention may also be used to treat SARS.

In another embodiment, the invention provides vaccine preparations,comprising the hSARS virus, including recombinant and chimeric forms ofsaid virus, or protein subunits of the virus. In a specific embodiment,the vaccine preparations of the present invention comprise live butattenuated hSARS virus with or without adjuvants. In another specificembodiment, the vaccine preparations of the invention comprise aninactivated or killed hSARS virus. Such attenuated or inactivatedviruses may be prepared by a series of passages of the virus through thehost cells or by preparing recombinant or chimeric forms of virus.Accordingly, the present invention further provides methods of preparingrecombinant or chimeric forms of hSARS. In another specific invention,the vaccine preparations of the present invention comprise a nucleicacid or fragment of the hSARS virus, e.g., the virus having accessionno. CCTCC-V200303, or nucleic acid molecules having the sequence of SEQID NO. 1, 11, 13, or 15, or a fragment thereof. In another embodiment,the invention provides vaccine preparations comprising one or morepolypeptides isolated from or produced from nucleic acid of hSARS virus,for example, of deposit accession no. CCTCC-V200303. In a specificembodiment, the vaccine preparations comprise a polypeptide of theinvention encoded by the nucleotide sequence of SEQ ID NO:1, 11, 13, 16,240, 737, 1108, 1590 or 1965, or a fragment thereof. In a specificembodiment, the vaccine preparations comprise polypeptides of theinvention as shown in FIGS. 11 (SEQ ID NOS:17-239, 241-736 and 738-1107)and 12 (SEQ ID NOS:1109-1589, 1591-1964 and 1966-2470) or encoded by thenucleotide sequence of SEQ ID NO:15, or a fragment thereof. Furthermore,the present invention provides methods for treating, ameliorating,managing or preventing SARS by administering the vaccine preparations orantibodies of the present invention alone or in combination withadjuvants, or other pharmaceutically acceptable excipients.

In another aspect, the present invention provides pharmaceuticalcompositions comprising anti-viral agents of the present invention and apharmaceutically acceptable carrier. In a specific embodiment, theanti-viral agent of the invention is an antibody that immunospecificallybinds hSARS virus or any hSARS epitope. In another specific embodiment,the anti-viral agent is a polypeptide or protein of the presentinvention or nucleic acid molecule of the invention. The invention alsoprovides kits containing a pharmaceutical composition of the presentinvention.

3.1 DEFINITIONS

The term “an antibody or an antibody fragment that immunospecificallybinds a polypeptide of the invention” as used herein refers to anantibody or a fragment thereof that immunospecifically binds to thepolypeptide encoded by the nucleotide sequence of SEQ ID NO:1, 11, 13 or15, or a fragment thereof, and does not non-specifically bind to otherpolypeptides. An antibody or a fragment thereof that immunospecificallybinds to the polypeptide of the invention may cross-react with otherantigens. Preferably, an antibody or a fragment thereof thatimmunospecifically binds to a polypeptide of the invention does notcross-react with other antigens. An antibody or a fragment thereof thatimmunospecifically binds to the polypeptide of the invention, can beidentified by, for example, immunoassays or other techniques known tothose skilled in the art.

An “isolated” or “purified” peptide or protein is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freeof chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations of a polypeptide/protein in which the polypeptide/proteinis separated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, a polypeptide/protein that issubstantially free of cellular material includes preparations of thepolypeptide/protein having less than about 30%, 20%, 10%, 5%, 2.5%, or1%, (by dry weight) of contaminating protein. When thepolypeptide/protein is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, or 5% of the volume of the proteinpreparation. When polypeptide/protein is produced by chemical synthesis,it is preferably substantially free of chemical precursors or otherchemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly, such preparations of the polypeptide/protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors orcompounds other than polypeptide/protein fragment of interest. In apreferred embodiment of the present invention, polypeptides/proteins areisolated or purified.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. In a preferred embodiment of the invention,nucleic acid molecules encoding polypeptides/proteins of the inventionare isolated or purified. The term “isolated” nucleic acid molecule doesnot include a nucleic acid that is a member of a library that has notbeen purified away from other library clones containing other nucleicacid molecules.

The term “portion” or “fragment” as used herein refers to a fragment ofa nucleic acid molecule containing at least about 25, 30, 35, 40, 45,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000,12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000,21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000,or more contiguous nucleic acids in length of the relevant nucleic acidmolecule and having at least one functional feature of the nucleic acidmolecule (or the encoded protein has one functional feature of theprotein encoded by the nucleic acid molecule); or a fragment of aprotein or a polypeptide containing at least 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 120, 140, 160, 180, 200,220, 240, 260, 280, 300, 320, 340, 360, 400, 500, 600, 700, 800, 900,1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,100, 4,200, 4,300,4,350, 4,360, 4,370, 4,380 amino acid residues in length of the relevantprotein or polypeptide and having at least one functional feature of theprotein or polypeptide.

The term “having a biological activity of the protein” or “havingbiological activities of the polypeptides of the invention” refers tothe characteristics of the polypeptides or proteins having a commonbiological activity similar or identical structural domain and/or havingsufficient amino acid identity to the polypeptide encoded by thenucleotide sequence of SEQ ID NO:1, 11, 13, 15, 16, 240, 737, 1108, 1590or 1965. Such common biological activities of the polypeptides of theinvention include antigenicity and immunogenicity.

The term “under stringent condition” refers to hybridization and washingconditions under which nucleotide sequences having at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%identity to each other remain hybridized to each other. Suchhybridization conditions are described in, for example but not limitedto, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6.; Basic Methods in Molecular Biology, ElsevierScience Publishing Co., Inc., N.Y. (1986), pp. 75-78, and 84-87; andMolecular Cloning, Cold Spring Harbor Laboratory, N.Y. (1982), pp.387-389, and are well known to those skilled in the art. A preferred,non-limiting example of stringent hybridization conditions ishybridization in 6× sodium chloride/sodium citrate (SSC), 0.5% SDS atabout 68° C. followed by one or more washes in 2×SSC, 0.5% SDS at roomtemperature. Another preferred, non-limiting example of stringenthybridization conditions is hybridization in 6×SSC at about 45° C.followed by one or more washes in 0.2×SSC, 0.1% SDS at about 50-65° C.

The term “variant” as used herein refers either to a naturally occurringgenetic mutant of hSARS or a recombinantly prepared variation of hSARSeach of which contain one or more mutations in its genome compared tothe hSARS of CCTCC-V200303. The term “variant” may also refers either toa naturally occurring variation of a given peptide or a recombinantlyprepared variation of a given peptide or protein in which one or moreamino acid residues have been modified by amino acid substitution,addition, or deletion.

4. DESCRIPTION OF THE FIGURES

FIG. 1 shows a partial DNA sequence (SEQ ID NO:1) and its deduced aminoacid sequence (SEQ ID NO:2) obtained from the SARS virus that has 57%homology to the RNA-dependent RNA polymerase protein of knownCoronaviruses.

FIG. 2 shows an electron micrograph of the novel hSARS virus that hassimilar morphological characteristics of coronaviruses.

FIG. 3 shows an immunofluorescent staining for IgG antibodies that arespecifically bound to the FrHK-4 cells infected with the novel humanrespiratory virus of Coronaviridae.

FIG. 4 shows an electron micrograph of ultra-centrifuged deposit ofhSARS virus that was grown in the cell culture and negatively stainedwith 3% potassium phospho-tungstate at pH 7.0.

FIG. 5A shows a thin-section electron micrograph of lung biopsy of apatient with SARS; and FIG. 5B shows a thin section electron micrographof hSARS-infected cells.

FIG. 6 shows the result of phylogenetic analysis for the partial proteinsequence (215 amino acids; SEQ ID NO:2) of the hSARS virus (GenBankaccession number AY268070). The phylogenetic tree is constructed by theneighbor-jointing method. The horizontal-line distance represents thenumber of sites at which the two sequences compared are different.Bootstrap values are deducted from 500 replicates.

FIG. 7A shows an amplification plot of fluorescence intensity againstthe PCR cycle in a real-time quantitative PCR assay that can detect ahSARS virus in samples quantitatively. The copy numbers of input plasmidDNA in the reactions are indicated. The X-axis denotes the cycle numberof a quantitative PCR assay and the Y-axis denotes the fluorescenceintensity (FI) over the background. FIG. 7B shows the result of amelting curve analysis of PCR products from clinical samples. Signalsfrom positive (+ve) samples, negative (−ve) samples and water control(water) are indicated. The X-axis denotes the temperature (° C.) and theY-axis denotes the fluorescence intensity (FI) over the background.

FIG. 8 shows another partial DNA sequence (SEQ ID NO:11) and its deducedamino acid sequence (SEQ ID NO:12) obtained from the SARS virus.

FIG. 9 shows yet another partial DNA sequence (SEQ ID NO:13) and itsdeduced amino acid sequence (SEQ ID NO:14) obtained from the SARS virus.

FIG. 10 shows the entire genomic DNA sequence (SEQ ID NO:15) of the SARSvirus.

FIG. 11 shows the deduced amino acid sequences obtained from SEQ IDNO:15 in three frames (see SEQ ID NOS:16, 240 and 737). An asterisk (*)indicates a stop codon which marks the end of a peptide. The first-frameamino acid sequences: SEQ ID NOS:17-239; the second-frame amino acidsequences: SEQ ID NOS:241-736; and the third-frame amino acid sequences:SEQ ID NO:738-1107.

FIG. 12 shows the deduced amino acid sequences obtained from thecomplement of SEQ ID NO:15 in three frames (see SEQ ID NOS:1108, 1590and 1965). An asterisk (*) indicates a stop codon which marks the end ofa peptide. The first-frame amino acid sequences: SEQ ID NOS:1109-1589;the second-frame amino acid sequences: SEQ ID NOS:1591-1964; and thethird-frame amino acid sequences: SEQ ID NO:1966-2470.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the isolated hSARS virus thatmorphologically and phylogenetically relates to known Coronaviruses. Ina specific embodiment, the isolated hSARS virus is that ofCCTCC-V200303. In another specific embodiment, the virus comprises anucleotide sequence of SEQ ID NO:1, 11, 13, and/or 15. In a specificembodiment, the present invention provides isolated nucleic acidmolecules of the hSARS virus, comprising, or, alternatively, consistingof the nucleotide sequence of SEQ ID NO:1, 11, 13, and/or 15, acomplement thereof or a portion thereof. In another specific embodiment,the invention provides isolated nucleic acid molecules which hybridizeunder stringent conditions, as defined herein, to a nucleic acidmolecule having the sequence of SEQ ID NO:1, 11, 13, or 15, or specificgenes of known member of Coronaviridae, or a complement thereof. Inanother specific embodiment, the invention provides isolatedpolypeptides or proteins that are encoded by a nucleic acid moleculecomprising a nucleotide sequence that is at least about 5, 10, 15, 20,25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, ormore contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1,or a complement thereof. In another specific embodiment, the inventionprovides isolated polypeptides or proteins that are encoded by a nucleicacid molecule comprising a nucleotide sequence that is at least about 5,10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150,1,200, or more contiguous nucleotides of the nucleotide sequence of SEQID NO:11, or a complement thereof. In yet another specific embodiment,the invention provides isolated polypeptides or proteins that areencoded by a nucleic acid molecule comprising a nucleotide sequence thatis at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300,350, 400, 450, 500, 550, 600, 650, 700, or more contiguous nucleotidesof the nucleotide sequence of SEQ ID NO:13, or a complement thereof. Inyet another specific embodiment, the invention provides isolatedpolypeptides or proteins that are encoded by a nucleic acid moleculecomprising or, alternatively consisting of a nucleotide sequence that isat least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,1,050, 1,100, 1,150, 1,200, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000,8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000,17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000,26,000, 27,000, 28,000, 29,000 or more contiguous nucleotides of thenucleotide sequence of SEQ ID NO:15, or a complement thereof. Thepolypeptides include those shown in FIGS. 11 (SEQ ID NOS:17-239, 241-736and 738-1107) and 12 (SEQ ID NOS:1109-1589, 1591-1964 and 1966-2470).The polypeptides or the proteins of the present invention preferablyhave one or more biological activities of the proteins encoded by thesequence of SEQ ID NO:1, 11, 13, 15, or the native viral proteinscontaining the amino acid sequences encoded by the sequence of SEQ IDNO:1, 11, 13, or 15, or those shown in FIGS. 11 (SEQ ID NOS:17-239,241-736 and 738-1107) and 12 (SEQ ID NOS:1109-1589, 1591-1964 and1966-2470).

The present invention also relates to a method for propagating the hSARSvirus in host cells.

The invention further relates to the use of the sequence information ofthe isolated virus for diagnostic and therapeutic methods. In a specificembodiment, the invention provides the entire nucleotide sequence ofhSARS virus, CCTCC-V200303, SEQ ID NO:15, or fragments, or complementthereof. Furthermore, the present invention relates to a nucleic acidmolecule that hybridizes any portion of the genome of the hSARS virus,CCTCC-V200303, SEQ ID NO:15, under the stringent conditions. In aspecific embodiment, the invention provides nucleic acid molecules whichare suitable for use as primers consisting of or comprising thenucleotide sequence of SEQ ID NO:1, 11, 13, or 15, or a complementthereof, or a portion thereof. In a non-limiting embodiment, theinvention provides the primers consisting of or comprising thenucleotide sequence of SEQ ID NOS:3 and/or 4. In another specificembodiment, the invention provides nucleic acid molecules which aresuitable for use as hybridization probes for the detection of nucleicacids encoding a polypeptide of the invention, consisting of orcomprising the nucleotide sequence of SEQ ID NO:1, 11, 13, or 15, acomplement thereof, or a portion thereof. The invention furtherencompasses chimeric or recombinant viruses or viral proteins encoded bysaid nucleotide sequences.

The invention further provides antibodies that specifically bind apolypeptide of the invention encoded by the nucleotide sequence of SEQID NO:1, 11, 13, 16, 240, 737, 1108, 1590 or 1965, or a fragmentthereof, or any hSARS epitope. The invention further provides antibodiesthat specifically bind the polypeptides of the invention encoded by thenucleotide sequence of SEQ ID NO:15, or a fragment thereof, or any hSARSepitope. Such antibodies include, but are not limited to polyclonal,monoclonal, bi-specific, multi-specific, human, humanized, chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,disulfide-linked Fvs, intrabodies and fragments containing either a VLor VH domain or even a complementary determining region (CDR) thatspecifically binds to a polypeptide of the invention.

In one embodiment, the invention provides methods for detecting thepresence, activity or expression of the hSARS virus of the invention ina biological material, such as cells, blood, saliva, urine, sputum,nasopharyngeal aspirates, and so forth. The presence of the hSARS virusin a sample can be determined by contacting the biological material withan agent which can detect directly or indirectly the presence of thehSARS virus. In a specific embodiment, the detection agents are theantibodies of the present invention. In another embodiment, thedetection agent is a nucleic acid of the present invention.

In another embodiment, the invention provides vaccine preparationscomprising the hSARS virus, including recombinant and chimeric forms ofsaid virus, or subunits of the virus. In a specific embodiment, thevaccine preparations comprise live but attenuated hSARS virus with orwithout pharmaceutically acceptable carriers, including adjuvants. Inanother specific embodiment, the vaccine preparations comprise aninactivated or killed hSARS virus with or without pharmaceuticallyacceptable carriers, including adjuvants.

The present invention further provides methods of preparing recombinantor chimeric forms of hSARS. In another specific invention, the vaccinepreparations of the present invention comprise one or more nucleic acidmolecules comprising or consisting of the sequence of SEQ ID NO. 1, 11,13, and/or, 15, or a fragment thereof. In another embodiment, theinvention provides vaccine preparations comprising one or morepolypeptides of the invention encoded by a nucleotide sequencecomprising or consisting of the nucleotide sequence of SEQ ID NO:1, 11,13, 16, 240, 737, 1108, 1590 and/or 1965, or a fragment thereof. Inanother embodiment, the invention provides vaccine preparationscomprising one or more polypeptides of the invention encoded by anucleotide sequence comprising or consisting of the nucleotide sequenceof SEQ ID NO:15, or a fragment thereof. Furthermore, the presentinvention provides methods for treating, ameliorating, managing, orpreventing SARS by administering the vaccine preparations or antibodiesof the present invention alone or in combination with antivirals [e.g.,amantadine, rimantadine, gancyclovir, acyclovir, ribavirin, penciclovir,oseltamivir, foscarnet zidovudine (AZT), didanosine (ddI), lamivudine(3TC), zalcitabine (ddC), stavudine (d4T), nevirapine, delavirdine,indinavir, ritonavir, vidarabine, nelfinavir, saquinavir, relenza,tamiflu, pleconaril, interferons, etc.], steroids and corticosteroidssuch as prednisone, cortisone, fluticasone and glucocorticoid,antibiotics, analgesics, bronchodialaters, or other treatments forrespiratory and/or viral infections.

Furthermore, the present invention provides pharmaceutical compositionscomprising anti-viral agents of the present invention and apharmaceutically acceptable carrier. The present invention also provideskits comprising pharmaceutical compositions of the present invention.

In another aspect, the present invention provides methods for screeninganti-viral agents that inhibit the infectivity or replication of hSARSvirus or variants thereof.

5.1 Recombinant and Chimeric hSARS Viruses

The present invention encompasses recombinant or chimeric virusesencoded by viral vectors derived from the genome of hSARS virus ornatural variants thereof. In a specific embodiment, a recombinant virusis one derived from the hSARS virus of deposit accession no.CCTCC-V200303. In a specific embodiment, the virus has a nucleotidesequence of SEQ ID NO:15. In another specific embodiment, a recombinantvirus is one derived from a natural variant of hSARS virus. A naturalvariant of hSARS has a sequence that is different from the genomicsequence (SEQ ID NO:15) of the hSARS virus, CCTCC-V200303, due to one ormore naturally occurred mutations, including, but not limited to, pointmutations, rearrangements, insertions, deletions etc., to the genomicsequence that may or may not result in a phenotypic change. Inaccordance with the present invention, a viral vector which is derivedfrom the genome of the hSARS virus, CCTCC-V200303, is one that containsa nucleic acid sequence that encodes at least a part of one ORF of thehSARS virus. In a specific embodiment, the ORF comprises or consists ofa nucleotide sequence of SEQ ID NO:1, 11 or 13, or a fragment thereof.In a specific embodiment, there are more than one ORF within thenucleotide sequence of SEQ ID NO:15 or a complement thereof, as shown inFIGS. 11 (SEQ ID NOS:16, 240 and 737) and 12 (SEQ ID NOS:1108, 1590 and1965), or a fragment thereof. In another embodiment, the polypeptideencoded by the ORF comprises or consists of an amino acid sequence ofSEQ ID NO:2, 12, or 14, or a fragment thereof, or shown in FIGS. 11 (SEQID NOS:17-239, 241-736 and 738-1107) and 12 (SEQ ID NOS:1109-1589,1591-1964 and 1966-2470), or a fragment thereof. In accordance with thepresent invention these viral vectors may or may not include nucleicacids that are non-native to the viral genome.

In another specific embodiment, a chimeric virus of the invention is arecombinant hSARS virus which further comprises a heterologousnucleotide sequence. In accordance with the invention, a chimeric virusmay be encoded by a nucleotide sequence in which heterologous nucleotidesequences have been added to the genome or in which endogenous or nativenucleotide sequences have been replaced with heterologous nucleotidesequences.

According to the present invention, the chimeric viruses are encoded bythe viral vectors of the invention which further comprise a heterologousnucleotide sequence. In accordance with the present invention a chimericvirus is encoded by a viral vector that may or may not include nucleicacids that are non-native to the viral genome. In accordance with theinvention a chimeric virus is encoded by a viral vector to whichheterologous nucleotide sequences have been added, inserted orsubstituted for native or non-native sequences. In accordance with thepresent invention, the chimeric virus may be encoded by nucleotidesequences derived from different strains or variants of hSARS virus. Inparticular, the chimeric virus is encoded by nucleotide sequences thatencode antigenic polypeptides derived from different strains or variantsof hSARS virus.

A chimeric virus may be of particular use for the generation ofrecombinant vaccines protecting against two or more viruses (Tao et al.,J. Virol. 72, 2955-2961; Durbin et al., 2000, J. Virol. 74, 6821-6831;Skiadopoulos et al., 1998, J. Virol. 72, 1762-1768 (1998); Teng et al.,2000, J. Virol. 74, 9317-9321). For example, it can be envisaged that avirus vector derived from the hSARS virus expressing one or moreproteins of variants of hSARS virus, or vice versa, will protect asubject vaccinated with such vector against infections by both thenative hSARS and the variant. Attenuated and replication-defectiveviruses may be of use for vaccination purposes with live vaccines as hasbeen suggested for other viruses. (See, PCT WO 02/057302, at pp. 6 and23, incorporated by reference herein).

In accordance with the present invention the heterologous sequence to beincorporated into the viral vectors encoding the recombinant or chimericviruses of the invention include sequences obtained or derived fromdifferent strains or variants of hSARS.

In certain embodiments, the chimeric or recombinant viruses of theinvention are encoded by viral vectors derived from viral genomeswherein one or more sequences, intergenic regions, termini sequences, orportions or entire ORF have been substituted with a heterologous ornon-native sequence. In certain embodiments of the invention, thechimeric viruses of the invention are encoded by viral vectors derivedfrom viral genomes wherein one or more heterologous sequences have beeninserted or added to the vector.

The selection of the viral vector may depend on the species of thesubject that is to be treated or protected from a viral infection. Ifthe subject is human, then an attenuated hSARS virus can be used toprovide the antigenic sequences.

In accordance with the present invention, the viral vectors can beengineered to provide antigenic sequences which confer protectionagainst infection by the hSARS and natural variants thereof. The viralvectors may be engineered to provide one, two, three or more antigenicsequences. In accordance with the present invention the antigenicsequences may be derived from the same virus, from different strains orvariants of the same type of virus, or from different viruses.

The expression products and/or recombinant or chimeric virions obtainedin accordance with the invention may advantageously be utilized invaccine formulations. The expression products and chimeric virions ofthe present invention may be engineered to create vaccines against abroad range of pathogens, including viral and bacterial antigens, tumorantigens, allergen antigens, and auto antigens involved in autoimmunedisorders. In particular, the chimeric virions of the present inventionmay be engineered to create vaccines for the protection of a subjectfrom infections with hSARS virus and variants thereof.

In certain embodiments, the expression products and recombinant orchimeric virions of the present invention may be engineered to createvaccines against a broad range of pathogens, including viral antigens,tumor antigens and autoantigens involved in autoimmune disorders. Oneway to achieve this goal involves modifying existing hSARS genes tocontain foreign sequences in their respective external domains. Wherethe heterologous sequences are epitopes or antigens of pathogens, thesechimeric viruses may be used to induce a protective immune responseagainst the disease agent from which these determinants are derived.

Thus, the present invention relates to the use of viral vectors andrecombinant or chimeric viruses to formulate vaccines against a broadrange of viruses and/or antigens. The present invention also encompassesrecombinant viruses comprising a viral vector derived from the hSARS orvariants thereof which contains sequences which result in a virus havinga phenotype more suitable for use in vaccine formulations, e.g.,attenuated phenotype or enhanced antigenicity. The mutations andmodifications can be in coding regions, in intergenic regions and in theleader and trailer sequences of the virus.

The invention provides a host cell comprising a nucleic acid or a vectoraccording to the invention. Plasmid or viral vectors containing thepolymerase components of hSARS virus are generated in prokaryotic cellsfor the expression of the components in relevant cell types (bacteria,insect cells, eukaryotic cells). Plasmid or viral vectors containingfull-length or partial copies of the hSARS genome will be generated inprokaryotic cells for the expression of viral nucleic acids in-vitro orin-vivo. The latter vectors may contain other viral sequences for thegeneration of chimeric viruses or chimeric virus proteins, may lackparts of the viral genome for the generation of replication defectivevirus, and may contain mutations, deletions or insertions for thegeneration of attenuated viruses. In addition, the present inventionprovides a host cell infected with hSARS virus, for example, of depositno. CCTCC-V200303.

Infectious copies of hSARS (being wild type, attenuated,replication-defective or chimeric) can be produced upon co-expression ofthe polymerase components according to the state-of-the-art technologiesdescribed above.

In addition, eukaryotic cells, transiently or stably expressing one ormore full-length or partial hSARS proteins can be used. Such cells canbe made by transfection (proteins or nucleic acid vectors), infection(viral vectors) or transduction (viral vectors) and may be useful forcomplementation of mentioned wild type, attenuated,replication-defective or chimeric viruses.

The viral vectors and chimeric viruses of the present invention may beused to modulate a subject's immune system by stimulating a humoralimmune response, a cellular immune response or by stimulating toleranceto an antigen. As used herein, a subject means: humans, primates,horses, cows, sheep, pigs, goats, dogs, cats, avian species and rodents.

5.2 Formulation of Vaccines and Antivirals

In a preferred embodiment, the invention provides a proteinaceousmolecule or hSARS virus specific viral protein or functional fragmentthereof encoded by a nucleic acid according to the invention. Usefulproteinaceous molecules are for example derived from any of the genes orgenomic fragments derivable from the virus according to the invention,including envelop protein (E protein), integral membrane protein (Mprotein), spike protein (S protein), nucleocapsid protein (N protein),hemaglutinin esterase (HE protein), and RNA-dependent RNA polymerase.Such molecules, or antigenic fragments thereof, as provided herein, arefor example useful in diagnostic methods or kits and in pharmaceuticalcompositions such as subunit vaccines. Particularly useful arepolypeptides encoded by the nucleotide sequence of SEQ ID NO:1, 11, 13,or 15, or as shown in FIGS. 11 (SEQ ID NOS:17-239, 241-736 and 738-1107)and 12 (SEQ ID NOS:1109-1589, 1591-1964 and 1966-2470), or antigenicfragments thereof for inclusion as antigen or subunit immunogen, butinactivated whole virus can also be used. Particularly useful are alsothose proteinaceous substances that are encoded by recombinant nucleicacid fragments of the hSARS genome, of course preferred are those thatare within the preferred bounds and metes of ORFs, in particular, foreliciting hSARS specific antibody or T cell responses, whether in vivo(e.g. for protective or therapeutic purposes or for providing diagnosticantibodies) or in vitro (e.g. by phage display technology or anothertechnique useful for generating synthetic antibodies).

The invention provides vaccine formulations for the prevention andtreatment of infections with hSARS virus. In certain embodiments, thevaccine of the invention comprises recombinant and chimeric viruses ofthe hSARS virus. In certain embodiments, the virus is attenuated.

In another embodiment of this aspect of the invention, inactivatedvaccine formulations may be prepared using conventional techniques to“kill” the chimeric viruses. Inactivated vaccines are “dead” in thesense that their infectivity has been destroyed. Ideally, theinfectivity of the virus is destroyed without affecting itsimmunogenicity. In order to prepare inactivated vaccines, the chimericvirus may be grown in cell culture or in the allantois of the chickembryo, purified by zonal ultracentrifugation, inactivated byformaldehyde or β-propiolactone, and pooled. The resulting vaccine isusually inoculated intramuscularly.

Inactivated viruses may be formulated with a suitable adjuvant in orderto enhance the immunological response. Such adjuvants may include butare not limited to mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols, polyanions;peptides; oil emulsions; and potentially useful human adjuvants such asBCG and Corynebacterium parvum.

In another aspect, the present invention also provides DNA vaccineformulations comprising a nucleic acid or fragment of the hSARS virus,e.g., the virus having accession no. CCTCC-V200303, or nucleic acidmolecules having the sequence of SEQ ID NO:1, 11, 13, or 15, or afragment thereof. In another specific embodiment, the DNA vaccineformulations of the present invention comprises a nucleic acid orfragment thereof encoding the antibodies which immunospecifically bindshSARS viruses. In DNA vaccine formulations, a vaccine DNA comprises aviral vector, such as that derived from the hSARS virus, bacterialplasmid, or other expression vector, bearing an insert comprising anucleic acid molecule of the present invention operably linked to one ormore control elements, thereby allowing expression of the vaccinatingproteins encoded by said nucleic acid molecule in a vaccinated subject.Such vectors can be prepared by recombinant DNA technology asrecombinant or chimeric viral vectors carrying a nucleic acid moleculeof the present invention (see also Section 5.1, supra).

Various heterologous vectors are described for DNA vaccinations againstviral infections. For example, the vectors described in the followingreferences may be used to express hSARS sequences instead of thesequences of the viruses or other pathogens described; in particular,vectors described for hepatitis B virus (Michel, M. L. et al., 1995,DAN-mediated immunization to the hepatitis B surface antigen in mice:Aspects of the humoral response mimic hepatitis B viral infection inhumans, Proc. Natl. Aca. Sci. USA 92:5307-5311; Davis, H. L. et al.,1993, DNA-based immunization induces continuous seretion of hepatitis Bsurface antigen and high levels of circulating antibody, Human Molec.Genetics 2:1847-1851), HIV virus (Wang, B. et al., 1993, Geneinoculation generates immune responses against human immunodeficiencyvirus type 1, Proc. Natl. Acad. Sci. USA 90:4156-4160; Lu, S. et al.,1996, Simian immunodeficiency virus DNA vaccine trial in macques, J.Virol. 70:3978-3991; Letvin, N. L. et al., 1997, Potent, protectiveanti-HIV immune responses generated by bimodal HIV envelope DNA plusprotein vaccination, Proc Natl Acad Sci USA. 94(17):9378-83), andinfluenza viruses (Robinson, H L et al., 1993, Protection against alethal influenza virus challenge by immunization with ahaemagglutinin-expressing plasmid DNA, Vaccine 11:957-960; Ulmer, J. B.et al., Heterologous protection against influenza by injection of DNAencoding a viral protein, Science 259:1745-1749), as well as bacterialinfections, such as tuberculosis (Tascon, R. E. et al., 1996,Vaccination against tuberculosis by DNA injection, Nature Med.2:888-892; Huygen, K. et al., 1996, Immunogenicity and protectiveefficacy of a tuberculosis DNA vaccine, Nature Med., 2:893-898), andparasitic infection, such as malaria (Sedegah, M., 1994, Protectionagainst malaria by immunization with plasmid DNA encodingcircumsporozoite protein, Proc. Natl. Acad. Sci. USA 91:9866-9870;Doolan, D. L. et al., 1996, Circumventing genetic restriction ofprotection against malaria with multigene DNA immunization: CD8+ Tcell-interferon δ, and nitric oxide-dependent immunity, J. Exper. Med.,1183:1739-1746).

Many methods may be used to introduce the vaccine formulations describedabove. These include, but are not limited to, oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, andintranasal routes. Alternatively, it may be preferable to introduce thechimeric virus vaccine formulation via the natural route of infection ofthe pathogen for which the vaccine is designed. The DNA vaccines of thepresent invention may be administered in saline solutions by injectionsinto muscle or skin using a syringe and needle (Wolff J. A. et al.,1990, Direct gene transfer into mouse muscle in vivo, Science247:1465-1468; Raz, E., 1994, Intradermal gene immunization: Thepossible role of DNA uptake in the induction of cellular immunity toviruses, Proc. Natl. Acd. Sci. USA 91:9519-9523). Another way toadminister DNA vaccines is called “gene gun” method, whereby microscopicgold beads coated with the DNA molecules of interest is fired into thecells (Tang, D. et al., 1992, Genetic immunization is a simple methodfor eliciting an immune response, Nature 356:152-154). For generalreviews of the methods for DNA vaccines, see Robinson, H. L., 1999, DNAvaccines: basic mechanism and immune responses (Review), Int. J. Mol.Med. 4(5):549-555; Barber, B., 1997, Introduction: Emerging vaccinestrategies, Seminars in Immunology 9(5):269-270; and Robinson, H. L. etal., 1997, DNA vaccines, Seminars in Immunology 9(5):271-283.

5.3 Attenuation of hSARS Virus or Variants Thereof

The hSARS virus or variants thereof of the invention can be geneticallyengineered to exhibit an attenuated phenotype. In particular, theviruses of the invention exhibit an attenuated phenotype in a subject towhich the virus is administered as a vaccine. Attenuation can beachieved by any method known to a skilled artisan. Without being boundby theory, the attenuated phenotype of the viruses of the invention canbe caused, e.g., by using a virus that naturally does not replicate wellin an intended host species, for example, by reduced replication of theviral genome, by reduced ability of the virus to infect a host cell, orby reduced ability of the viral proteins to assemble to an infectiousviral particle relative to the wild type strain of the virus.

The attenuated phenotypes of hSARS virus or variants thereof can betested by any method known to the artisan. A candidate virus can, forexample, be tested for its ability to infect a host or for the rate ofreplication in a cell culture system. In certain embodiments, growthcurves at different temperatures are used to test the attenuatedphenotype of the virus. For example, an attenuated virus is able to growat 35° C., but not at 39° C. or 40° C. In certain embodiments, differentcell lines can be used to evaluate the attenuated phenotype of thevirus. For example, an attenuated virus may only be able to grow inmonkey cell lines but not the human cell lines, or the achievable virustiters in different cell lines are different for the attenuated virus.In certain embodiments, viral replication in the respiratory tract of asmall animal model, including but not limited to, hamsters, cotton rats,mice and guinea pigs, is used to evaluate the attenuated phenotypes ofthe virus. In other embodiments, the immune response induced by thevirus, including but not limited to, the antibody titers (e.g., assayedby plaque reduction neutralization assay or ELISA) is used to evaluatethe attenuated phenotypes of the virus. In a specific embodiment, theplaque reduction neutralization assay or ELISA is carried out at a lowdose. In certain embodiments, the ability of the hSARS virus to elicitpathological symptoms in an animal model can be tested. A reducedability of the virus to elicit pathological symptoms in an animal modelsystem is indicative of its attenuated phenotype. In a specificembodiment, the candidate viruses are tested in a monkey model for nasalinfection, indicated by mucous production.

The viruses of the invention can be attenuated such that one or more ofthe functional characteristics of the virus are impaired. In certainembodiments, attenuation is measured in comparison to the wild typestrain of the virus from which the attenuated virus is derived. In otherembodiments, attenuation is determined by comparing the growth of anattenuated virus in different host systems. Thus, for a non-limitingexample, hSARS virus or a variant thereof is said to be attenuated whengrown in a human host if the growth of the hSARS or variant thereof inthe human host is reduced compared to the non-attenuated hSARS orvariant thereof.

In certain embodiments, the attenuated virus of the invention is capableof infecting a host, is capable of replicating in a host such thatinfectious viral particles are produced. In comparison to the wild typestrain, however, the attenuated strain grows to lower titers or growsmore slowly. Any technique known to the skilled artisan can be used todetermine the growth curve of the attenuated virus and compare it to thegrowth curve of the wild type virus.

In certain embodiments, the attenuated virus of the invention (e.g., arecombinant or chimeric hSARS) cannot replicate in human cells as wellas the wild type virus (e.g., wild type hSARS) does. However, theattenuated virus can replicate well in a cell line that lack interferonfunctions, such as Vero cells.

In other embodiments, the attenuated virus of the invention is capableof infecting a host, of replicating in the host, and of causing proteinsof the virus of the invention to be inserted into the cytoplasmicmembrane, but the attenuated virus does not cause the host to producenew infectious viral particles. In certain embodiments, the attenuatedvirus infects the host, replicates in the host, and causes viralproteins to be inserted in the cytoplasmic membrane of the host with thesame efficiency as the wild type hSARS. In other embodiments, theability of the attenuated virus to cause viral proteins to be insertedinto the cytoplasmic membrane into the host cell is reduced compared tothe wild type virus. In certain embodiments, the ability of theattenuated hSARS virus to replicate in the host is reduced compared tothe wild type virus. Any technique known to the skilled artisan can beused to determine whether a virus is capable of infecting a mammaliancell, of replicating within the host, and of causing viral proteins tobe inserted into the cytoplasmic membrane of the host.

In certain embodiments, the attenuated virus of the invention is capableof infecting a host. In contrast to the wild type hSARS, however, theattenuated hSARS cannot be replicated in the host. In a specificembodiment, the attenuated hSARS virus can infect a host and can causethe host to insert viral proteins in its cytoplasmic membranes, but theattenuated virus is incapable of being replicated in the host. Anymethod known to the skilled artisan can be used to test whether theattenuated hSARS has infected the host and has caused the host to insertviral proteins in its cytoplasmic membranes.

In certain embodiments, the ability of the attenuated virus to infect ahost is reduced compared to the ability of the wild type virus to infectthe same host. Any technique known to the skilled artisan can be used todetermine whether a virus is capable of infecting a host.

In certain embodiments, mutations (e.g., missense mutations) areintroduced into the genome of the virus, for example, into the sequenceof SEQ ID NO:1, 11, 13, or 15, or to generate a virus with an attenuatedphenotype. Mutations (e.g., missense mutations) can be introduced intothe structural genes and/or regulatory genes of the hSARS. Mutations canbe additions, substitutions, deletions, or combinations thereof. Suchvariant of hSARS can be screened for a predicted functionality, such asinfectivity, replication ability, protein synthesis ability, assemblingability, as well as cytopathic effect in cell cultures. In a specificembodiment, the missense mutation is a cold-sensitive mutation. Inanother embodiment, the missense mutation is a heat-sensitive mutation.In another embodiment, the missense mutation prevents a normalprocessing or cleavage of the viral proteins.

In other embodiments, deletions are introduced into the genome of thehSARS virus, which result in the attenuation of the virus.

In certain embodiments, attenuation of the virus is achieved byreplacing a gene of the wild type virus with a gene of a virus of adifferent species, of a different subgroup, or of a different variant.In another aspect, attenuation of the virus is achieved by replacing oneor more specific domains of a protein of the wild type virus withdomains derived from the corresponding protein of a virus of a differentspecies. In certain other embodiments, attenuation of the virus isachieved by deleting one or more specific domains of a protein of thewild type virus.

When a live attenuated vaccine is used, its safety must also beconsidered. The vaccine must not cause disease. Any techniques known inthe art that can make a vaccine safe may be used in the presentinvention. In addition to attenuation techniques, other techniques maybe used. One non-limiting example is to use a soluble heterologous genethat cannot be incorporated into the virion membrane. For example, asingle copy of the soluble version of a viral transmembrane proteinlacking the transmembrane and cytosolic domains thereof, can be used.

Various assays can be used to test the safety of a vaccine. For example,sucrose gradients and neutralization assays can be used to test thesafety. A sucrose gradient assay can be used to determine whether aheterologous protein is inserted in a virion. If the heterologousprotein is inserted in the virion, the virion should be tested for itsability to cause symptoms in an appropriate animal model since the virusmay have acquired new, possibly pathological, properties.

5.4 Adjuvants and Carrier Molecules

hSARS-associated antigens are administered with one or more adjuvants.In one embodiment, the hSARS-associated antigen is administered togetherwith a mineral salt adjuvants or mineral salt gel adjuvant. Such mineralsalt and mineral salt gel adjuvants include, but are not limited to,aluminum hydroxide (ALHYDROGEL, REHYDRAGEL), aluminum phosphate gel,aluminum hydroxyphosphate (ADJU-PHOS), and calcium phosphate.

In another embodiment, hSARS-associated antigen is administered with animmunostimulatory adjuvant. Such class of adjuvants, include, but arenot limited to, cytokines (e.g., interleukin-2, interleukin-7,interleukin-12, granulocyte-macrophage colony stimulating factor(GM-CSF), interferon-γ interleukin-1β (IL-1β), and IL-1β peptide orSclavo Peptide), cytokine-containing liposomes, triterpenoid glycosidesor saponins (e.g., QuilA and QS-21, also sold under the trademarkSTIMULON, ISCOPREP), Muramyl Dipeptide (MDP) derivatives, such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (Threonyl-MDP, sold under thetrademark TERMURTIDE), GMDP,N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine, muramyl tripeptide phosphatidylethanolamine(MTP-PE), unmethylated CpG dinucleotides and oligonucleotides, such asbacterial DNA and fragments thereof, LPS, monophosphoryl Lipid A (3D-MLAsold under the trademark MPL), and polyphosphazenes.

In another embodiment, the adjuvant used is a particular adjuvant,including, but not limited to, emulsions, e.g., Freund's CompleteAdjuvant, Freund's Incomplete Adjuvant, squalene or squalaneoil-in-water adjuvant formulations, such as SAF and MF59, e.g., preparedwith block-copolymers, such as L-121 (polyoxypropylene/polyoxyetheylene)sold under the trademark PLURONIC L-121, Liposomes, Virosomes,cochleates, and immune stimulating complex, which is sold under thetrademark ISCOM.

In another embodiment, a microparticular adjuvant is used.,Microparticulare adjuvants include, but are not limited to biodegradableand biocompatible polyesters, homo- and copolymers of lactic acid (PLA)and glycolic acid (PGA), poly(lactide-co-glycolides) (PLGA)microparticles, polymers that self-associate into particulates(poloxamer particles), soluble polymers (polyphosphazenes), andvirus-like particles (VLPs) such as recombinant protein particulates,e.g., hepatitis B surface antigen (HbsAg).

Yet another class of adjuvants that may be used include mucosaladjuvants, including but not limited to heat-labile enterotoxin fromEscherichia coli (LT), cholera holotoxin (CT) and cholera Toxin BSubunit (CTB) from Vibrio cholerae, mutant toxins (e.g., LTK63 andLTR72), microparticles, and polymerized liposomes.

In other embodiments, any of the above classes of adjuvants may be usedin combination with each other or with other adjuvants. For example,non-limiting examples of combination adjuvant preparations that can beused to administer the hSARS-associated antigens of the inventioninclude liposomes containing immunostimulatory protein, cytokines, orT-cell and/or B-cell peptides, or microbes with or without entrappedIL-2 or microparticles containing enterotoxin. Other adjuvants known inthe art are also included within the scope of the invention (see VaccineDesign: The Subunit and Adjuvant Approach, Chap. 7, Michael F. Powelland Mark J. Newman (eds.), Plenum Press, New York, 1995, which isincorporated herein in its entirety).

The effectiveness of an adjuvant may be determined by measuring theinduction of antibodies directed against an immunogenic polypeptidecontaining a hSARS polypeptide epitope, the antibodies resulting fromadministration of this polypeptide in vaccines which are also comprisedof the various adjuvants.

The polypeptides may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts include the acid additionalsalts (formed with free amino groups of the peptide) and which areformed with inorganic acids, such as, for example, hydrochloric orphosphoric acids, or organic acids such as acetic, oxalic, tartaric,maleic, and the like. Salts formed with free carboxyl groups may also bederived from inorganic bases, such as, for example, sodium potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine and the like.

The vaccines of the invention may be multivalent or univalent.Multivalent vaccines are made from recombinant viruses that direct theexpression of more than one antigen.

Many methods may be used to introduce the vaccine formulations of theinvention; these include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasalroutes, and via scarification (scratching through the top layers ofskin, e.g., using a bifurcated needle).

The patient to which the vaccine is administered is preferably a mammal,most preferably a human, but can also be a non-human animal includingbut not limited to cows, horses, sheep, pigs, fowl (e.g., chickens),goats, cats, dogs, hamsters, mice and rats.

5.5 Preparation of Antibodies

Antibodies which specifically recognize a polypeptide of the invention,such as, but not limited to, polypeptides comprising the sequence of SEQID NO:2, 12, and 14, and polypeptides as shown in FIGS. 11 (SEQ IDNOS:17-239, 241-736 and 738-1107) and 12 (SEQ ID NOS:1109-1589,1591-1964 and 1966-2470), or hSARS epitope or antigen-binding fragmentsthereof can be used for detecting, screening, and isolating thepolypeptide of the invention or fragments thereof, or similar sequencesthat might encode similar enzymes from the other organisms. For example,in one specific embodiment, an antibody which immunospecifically bindshSARS epitope, or a fragment thereof, can be used for various in vitrodetection assays, including enzyme-linked immunosorbent assays (ELISA),radioimmunoassays, Western blot, etc., for the detection of apolypeptide of the invention or, preferably, hSARS, in samples, forexample, a biological material, including cells, cell culture media(e.g., bacterial cell culture media, mammalian cell culture media,insect cell culture media, yeast cell culture media, etc.), blood,plasma, serum, tissues, sputum, naseopharyngeal aspirates, etc.

Antibodies specific for a polypeptide of the invention or any epitope ofhSARS may be generated by any suitable method known in the art.Polyclonal antibodies to an antigen-of-interest, for example, the hSARSvirus from deposit no. CCTCC-V200303, or comprises a nucleotide sequenceof SEQ ID NO:15, can be produced by various procedures well known in theart. For example, an antigen can be administered to various host animalsincluding, but not limited to, rabbits, mice, rats, etc., to induce theproduction of antisera containing polyclonal antibodies specific for theantigen. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, and include but are not limitedto, Freund's (complete and incomplete) adjuvant, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful adjuvants for humanssuch as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Suchadjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (both of which areincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CHI region and the hinge region of the heavy chain.

The antibodies of the invention or fragments thereof can be alsoproduced by any method known in the art for the synthesis of antibodies,in particular, by chemical synthesis or preferably, by recombinantexpression techniques.

The nucleotide sequence encoding an antibody may be obtained from anyinformation available to those skilled in the art (i.e., from Genbank,the literature, or by routine cloning and sequence analysis). If a clonecontaining a nucleic acid encoding a particular antibody or anepitope-binding fragment thereof is not available, but the sequence ofthe antibody molecule or epitope-binding fragment thereof is known, anucleic acid encoding the immunoglobulin may be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody) by PCR amplificationusing synthetic primers hybridizable to the 3′ and 5′ ends of thesequence or by cloning using an oligonucleotide probe specific for theparticular gene sequence to identify, e.g., a cDNA clone from a cDNAlibrary that encodes the antibody. Amplified nucleic acids generated byPCR may then be cloned into replicable cloning vectors using any methodwell known in the art.

Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al., supra;and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties), to generate antibodies having a different aminoacid sequence by, for example, introducing amino acid substitutions,deletions, and/or insertions into the epitope-binding domain regions ofthe antibodies or any portion of antibodies which may enhance or reducebiological activities of the antibodies.

Recombinant expression of an antibody requires construction of anexpression vector containing a nucleotide sequence that encodes theantibody. Once a nucleotide sequence encoding an antibody molecule or aheavy or light chain of an antibody, or portion thereof has beenobtained, the vector for the production of the antibody molecule may beproduced by recombinant DNA technology using techniques well known inthe art as discussed in the previous sections. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing antibody coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. The nucleotide sequenceencoding the heavy-chain variable region, light-chain variable region,both the heavy-chain and light-chain variable regions, anepitope-binding fragment of the heavy- and/or light-chain variableregion, or one or more complementarity determining regions (CDRs) of anantibody may be cloned into such a vector for expression. Thus-preparedexpression vector can be then introduced into appropriate host cells forthe expression of the antibody. Accordingly, the invention includes hostcells containing a polynucleotide encoding an antibody specific for thepolypeptides of the invention or fragments thereof.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides or different selectablemarkers to ensure maintenance of both plasmids. Alternatively, a singlevector may be used which encodes, and is capable of expressing, bothheavy and light chain polypeptides. In such situations, the light chainshould be placed before the heavy chain to avoid an excess of toxic freeheavy chain (Proudfoot, Nature, 322:52, 1986; and Kohler, Proc. Natl.Acad. Sci. USA, 77:2 197, 1980). The coding sequences for the heavy andlight chains may comprise cDNA or genomic DNA.

In another embodiment, antibodies can also be generated using variousphage display methods known in the art. In phage display methods,functional antibody domains are displayed on the surface of phageparticles which carry the polynucleotide sequences encoding them. In aparticular embodiment, such phage can be utilized to display antigenbinding domains, such as Fab and Fv or disulfide-bond stabilized Fv,expressed from a repertoire or combinatorial antibody library (e.g.,human or murine). Phage expressing an antigen binding domain that bindsthe antigen of interest can be selected or identified with antigen,e.g., using labeled antigen or antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage, including fd and M13. The antigen binding domains are expressedas a recombinantly fused protein to either the phage gene III or geneVIII protein. Examples of phage display methods that can be used to makethe immunoglobulins, or fragments thereof, of the present inventioninclude those disclosed in Brinkman et al., J. Immunol. Methods,182:41-50, 1995; Ames et al., J. Immunol. Methods, 184:177-186, 1995;Kettleborough et al., Eur. J. Immunol., 24:952-958, 1994; Persic et al.,Gene, 187:9-18, 1997; Burton et al., Advances in Immunology, 57:191-280,1994; PCT application No. PCT/GB91/01134; PCT publications WO 90/02809;WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which isincorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab′)₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT publication WO92/22324; Mullinax et al., BioTechniques, 12(6):864-869, 1992; and Sawaiet al., AJRI, 34:26-34, 1995; and Better et al., Science, 240:1041-1043,1988 (each of which is incorporated by reference in its entirety).Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu etal., PNAS, 90:7995-7999, 1993; and Skerra et al., Science,240:1038-1040, 1988.

Once an antibody molecule of the invention has been produced by anymethods described above, it may then be purified by any method known inthe art for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A or Protein G purification, andsizing column chromatography), centrifugation, differential solubility,or by any other standard techniques for the purification of proteins.Further, the antibodies of the present invention or fragments thereofmay be fused to heterologous polypeptide sequences described herein orotherwise known in the art to facilitate purification.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a constant region derived from a humanimmunoglobulin. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science, 229:1202, 1985; Oi et al.,BioTechniques, 4:214 1986; Gillies et al., J. Immunol. Methods,125:191-202, 1989; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,which are incorporated herein by reference in their entireties.Humanized antibodies are antibody molecules from non-human species thatbind the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature, 332:323, 1988, which areincorporated herein by reference in their entireties. Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology, 28(4/5):489-498,1991; Studnicka et al., Protein Engineering, 7(6):805-814, 1994; Roguskaet al., Proc Natl. Acad. Sci. USA, 91:969-973, 1994), and chainshuffling (U.S. Pat. No. 5,565,332), all of which are herebyincorporated by reference in their entireties.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For an overview of thistechnology for producing human antibodies, see Lonberg and Huszar, Int.Rev. Immunol., 13:65-93, 1995. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Abgenix, Inc. (Fremont,Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology,12:899-903, 1988).

Antibodies fused or conjugated to heterologous polypeptides may be usedin in vitro immunoassays and in purification methods (e.g., affinitychromatography) well known in the art. See e.g., PCT publication NumberWO 93/21232; EP 439,095; Naramura et al., Immunol. Lett., 39:91-99,1994; U.S. Pat. No. 5,474,981; Gillies et al., PNAS, 89:1428-1432, 1992;and Fell et al., J. Immunol., 146:2446-2452, 1991, which areincorporated herein by reference in their entireties.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the polypeptidesof the invention or fragments, derivatives, analogs, or variantsthereof, or similar molecules having the similar enzymatic activities asthe polypeptide of the invention. Such solid supports include, but arenot limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene.

5.6 Pharmaceutical Compositions and Kits

The present invention encompasses pharmaceutical compositions comprisinganti-viral agents of the present invention. In a specific embodiment,the anti-viral agent is an antibody which immunospecifically binds andneutralize the hSARS virus or variants thereof, or any proteins derivedtherefrom (see Section 5.5). In another specific embodiment, theanti-viral agent is a polypeptide or nucleic acid molecule of theinvention (see, for example, Sections 5.1 and 5.2). The pharmaceuticalcompositions have utility as an anti-viral prophylactic agent and may beadministered to a subject where the subject has been exposed or isexpected to be exposed to a virus.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu and Wu, 1987, J. Biol. Chem. 262:4429 4432). Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Ina preferred embodiment, it may be desirable to introduce thepharmaceutical compositions of the invention into the lungs by anysuitable route. Pulmonary administration can also be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, by means of nasal spray, or bymeans of an implant, said implant being of a porous, non porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. In one embodiment, administration can be by direct injectionat the site (or former site) infected tissues.

In another embodiment, the pharmaceutical composition can be deliveredin a vesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.).

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; and Saudek et al., 1989,N. Engl. J. Med. 321:574). In another embodiment, polymeric materialscan be used (see Medical Applications of Controlled Release, Langer andWise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, i.e., thelung, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of an live attenuated, inactivated orkilled hSARS virus, or recombinant or chimeric hSARS virus, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the pharmaceuticalcomposition is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. The formulation should suitthe mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2ethylamino ethanol, histidine, procaine, etc.

The amount of the pharmaceutical composition of the invention which willbe effective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20 500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. In apreferred embodiment, the kit contains an anti-viral agent of theinvention, e.g., an antibody specific for the polypeptides encoded by anucleotide sequence of SEQ ID NO:1, 11, 13, or 15, or as shown in FIGS.11 (SEQ ID NOS:17-239, 241-736 and 738-1107) and 12 (SEQ IDNOS:1109-1589, 1591-1964 and 1966-2470), or any hSARS epitope, or apolypeptide or protein of the present invention, or a nucleic acidmolecule of the invention, alone or in combination with adjuvants,antivirals, antibiotics, analgesic, bronchodialaters, or otherpharmaceutically acceptable excipients.

The present invention further encompasses kits comprising a containercontaining a pharmaceutical composition of the present invention andinstructions to for use.

5.7 Detection Assays

The present invention provides a method for detecting an antibody, whichimmunospecifically binds to the hSARS virus, in a biological sample, forexample blood, serum, plasma, saliva, urine, etc., from a patientsuffering from SARS. In a specific embodiment, the method comprisingcontacting the sample with the hSARS virus, for example, of deposit no.CCTCC-V200303, or having a genomic nucleic acid sequence of SEQ IDNO:15, directly immobilized on a substrate and detecting the virus-boundantibody directly or indirectly by a labeled heterologous anti-isotypeantibody. In another specific embodiment, the sample is contacted with ahost cell which is infected by the hSARS virus, for example, of depositno. CCTCC-V200303, or having a genomic nucleic acid sequence of SEQ IDNO:15, and the bound antibody can be detected by immunofluorescent assayas described in Section 6.5, infra.

An exemplary method for detecting the presence or absence of apolypeptide or nucleic acid of the invention in a biological sampleinvolves obtaining a biological sample from various sources andcontacting the sample with a compound or an agent capable of detectingan epitope or nucleic acid (e.g., mRNA, genomic DNA) of the hSARS virussuch that the presence of the hSARS virus is detected in the sample. Apreferred agent for detecting hSARS mRNA or genomic RNA of the inventionis a labeled nucleic acid probe capable of hybridizing to mRNA orgenomic RNA encoding a polypeptide of the invention. The nucleic acidprobe can be, for example, a nucleic acid molecule comprising orconsisting of the nucleotide sequence of SEQ ID NO:1, 11, 13, or 15, ora portion thereof, such as an oligonucleotide of at least 15, 20, 25,30, 50, 100, 250, 500, 750, 1,000 or more contiguous nucleotides inlength and sufficient to specifically hybridize under stringentconditions to a hSARS mRNA or genomic RNA.

In another preferred specific embodiment, the presence of hSARS virus isdetected in the sample by an reverse transcription polymerase chainreaction (RT-PCR) using the primers that are constructed based on apartial nucleotide sequence of the genome of hSARS virus, for example,that of deposit accession no. CCTCC-V200303, or having a genomic nucleicacid sequence of SEQ ID NO:15, or based on a nucleotide sequence of SEQID NO:1, 11, 13, or 15. In a non-limiting specific embodiment, preferredprimers to be used in a RT-PCR method are: 5′-TACACACCTCAGC-GTTG-3′ (SEQID NO:3) and 5′-CACGAACGTGACG-AAT-3′ (SEQ ID NO:4), in the presence of2.5 mM MgCl₂ and the thermal cycles are, for example, but not limitedto, 94° C. for 8 min followed by 40 cycles of 94° C. for 1 min, 50° C.for 1 min, 72° C. for 1 min (also see Section 6.7, infra). In morepreferred specific embodiment, the present invention provides areal-time quantitative PCR assay to detect the presence of hSARS virusin a biological sample by subjecting the cDNA obtained by reversetranscription of the extracted total RNA from the sample to PCRreactions using the specific primers, such as those having nucleotidesequences of SEQ ID NOS:3 and 4, and a fluorescence dye, such as SYBR®Green I, which fluoresces when bound non-specifically to double-strandedDNA. The fluorescence signals from these reactions are captured at theend of extension steps as PCR product is generated over a range of thethermal cycles, thereby allowing the quantitative determination of theviral load in the sample based on an amplification plot (see Section6.7, infra).

A preferred agent for detecting hSARS is an antibody that specificallybinds a polypeptide of the invention or any hSARS epitope, preferably anantibody with a detectable label. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,Fab or F(ab′)₂) can be used.

The term “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. Thedetection method of the invention can be used to detect mRNA, protein(or any epitope), or genomic RNA in a sample in vitro as well as invivo. For example, in vitro techniques for detection of mRNA includenorthern hybridizations, in situ hybridizations, RT-PCR, and RNaseprotection. In vitro techniques for detection of an epitope of hSARSinclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of genomic RNA include northern hybridizations, RT-PCT, andRNase protection. Furthermore, in vivo techniques for detection of hSARSinclude introducing into a subject organism a labeled antibody directedagainst the polypeptide. For example, the antibody can be labeled with aradioactive marker whose presence and location in the subject organismcan be detected by standard imaging techniques, includingautoradiography.

In a specific embodiment, the methods further involve obtaining acontrol sample from a control subject, contacting the control samplewith a compound or agent capable of detecting hSARS, e.g., a polypeptideof the invention or mRNA or genomic RNA encoding a polypeptide of theinvention, such that the presence of hSARS or the polypeptide or mRNA orgenomic RNA encoding the polypeptide is detected in the sample, andcomparing the absence of hSARS or the polypeptide or mRNA or genomic RNAencoding the polypeptide in the control sample with the presence ofhSARS, or the polypeptide or mRNA or genomic DNA encoding thepolypeptide in the test sample.

The invention also encompasses kits for detecting the presence of hSARSor a polypeptide or nucleic acid of the invention in a test sample. Thekit, for example, can comprise a labeled compound or agent capable ofdetecting hSARS or the polypeptide or a nucleic acid molecule encodingthe polypeptide in a test sample and, in certain embodiments, a meansfor determining the amount of the polypeptide or mRNA in the sample(e.g., an antibody which binds the polypeptide or an oligonucleotideprobe which binds to DNA or mRNA encoding the polypeptide). Kits canalso include instructions for use.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide of the invention or hSARS epitope; and, optionally, (2) asecond, different antibody which binds to either the polypeptide or thefirst antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptide of theinvention or to a sequence within the hSARS genome or (2) a pair ofprimers useful for amplifying a nucleic acid molecule containing anhSARS sequence. The kit can also comprise, e.g., a buffering agent, apreservative, or a protein stabilizing agent. The kit can also comprisecomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for use.

5.8 Screening Assays to Identify Anti-Viral Agents

The invention provides methods for the identification of a compound thatinhibits the ability of hSARS virus to infect a host or a host cell. Incertain embodiments, the invention provides methods for theidentification of a compound that reduces the ability of hSARS virus toreplicate in a host or a host cell. Any technique well-known to theskilled artisan can be used to screen for a compound that would abolishor reduce the ability of hSARS virus to infect a host and/or toreplicate in a host or a host cell.

In certain embodiments, the invention provides methods for theidentification of a compound that inhibits the ability of hSARS virus toreplicate in a mammal or a mammalian cell. More specifically, theinvention provides methods for the identification of a compound thatinhibits the ability of hSARS virus to infect a mammal or a mammaliancell. In certain embodiments, the invention provides methods for theidentification of a compound that inhibits the ability of hSARS virus toreplicate in a mammalian cell. In a specific embodiment, the mammaliancell is a human cell.

In another embodiment, a cell is contacted with a test compound andinfected with the hSARS virus. In certain embodiments, a control cultureis infected with the hSARS virus in the absence of a test compound. Thecell can be contacted with a test compound before, concurrently with, orsubsequent to the infection with the hSARS virus. In a specificembodiment, the cell is a mammalian cell. In an even more specificembodiment, the cell is a human cell. In certain embodiments, the cellis incubated with the test compound for at least 1 minute, at least 5minutes at least 15 minutes, at least 30 minutes, at least 1 hour, atleast 2 hours, at least 5 hours, at least 12 hours, or at least 1 day.The titer of the virus can be measured at any time during the assay. Incertain embodiments, a time course of viral growth in the culture isdetermined. If the viral growth is inhibited or reduced in the presenceof the test compound, the test compound is identified as being effectivein inhibiting or reducing the growth or infection of the hSARS virus. Ina specific embodiment, the compound that inhibits or reduces the growthof the hSARS virus is tested for its ability to inhibit or reduce thegrowth rate of other viruses to test its specificity for the hSARSvirus.

In one embodiment, a test compound is administered to a model animal andthe model animal is infected with the hSARS virus. In certainembodiments, a control model animal is infected with the hSARS viruswithout the administration of a test compound. The test compound can beadministered before, concurrently with, or subsequent to the infectionwith the hSARS virus. In a specific embodiment, the model animal is amammal. In an even more specific embodiment, the model animal can be,but is not limited to, a cotton rat, a mouse, or a monkey. The titer ofthe virus in the model animal can be measured at any time during theassay. In certain embodiments, a time course of viral growth in theculture is determined. If the viral growth is inhibited or reduced inthe presence of the test compound, the test compound is identified asbeing effective in inhibiting or reducing the growth or infection of thehSARS virus. In a specific embodiment, the compound that inhibits orreduces the growth of the hSARS in the model animal is tested for itsability to inhibit or reduce the growth rate of other viruses to testits specificity for the hSARS virus.

6. EXAMPLES

The following examples illustrate the isolation and identification ofthe novel hSARS virus. These examples should not be construed aslimiting.

METHODS AND RESULTS

As a general reference, Wiedbrauk D L & Johnston S L G. (Manual ofClinical Virology, Raven Press, New York, 1993) was used.

6.1 Clinical Subjects

The study included all 50 patients who fitted a modified World HealthOrganization (WHO) definition of SARS and were admitted to 2 acuteregional hospitals in Hong Kong Special Administrative Region (HKSAR)between Feb. 26 to Mar. 26, 2003 (WHO. Severe acute respiratory syndrome(SARS) Weekly Epidemiol Rec. 2003; 78: 81-83). A lung biopsy from anadditional patient, who had typical SARS and was admitted to a thirdhospital, was also included in the study. Briefly, the case definitionfor SARS was: (i) fever of 38° C. or more; (ii) cough or shortness ofbreath; (iii) new pulmonary infiltrates on chest radiograph; and (iv)either a history of exposure to a patient with SARS or absence ofresponse to empirical antimicrobial coverage for typical and atypicalpneumonia (beta-lactams and macrolides, fluoroquinolones ortetracyclines).

Nasopharyngeal aspirates and serum samples were collected from allpatients. Paired acute and convalescent sera and feces were availablefrom some patients. Lung biopsy tissue from one patient was processedfor a viral culture, RT-PCR, routine histopathological examination, andelectron microscopy. Nasopharyngeal aspirates, feces and sera submittedfor microbiological investigation of other diseases were included in thestudy under blinding and served as controls.

The medical records were reviewed retrospectively by the attendingphysicians and clinical microbiologists. Routine hematological,biochemical and microbiological examinations, including bacterialculture of blood and sputum, serological study and collection ofnasopharyngeal aspirates for virological tests, were carried out.

6.2 Cell Line

FRhK-4 (fetal rhesus monkey kidney) cells were maintained in minimalessential medium (MEM) with 1% fetal calf serum, 1% streptomycin andpenicillin, 0.2% nystatin and 0.05% garamycin.

6.3 Viral Infection

Two-hundred μl of clinical (nasopharyngeal aspirates) samples, from twopatients (see the Result section, infra), in virus transport medium wereused to infect FRhk-4 cells. The inoculated cells were incubated at 37°C. for 1 hour. One ml of MEM containing 1 μg trypsin was then added tothe culture and the infected cells were incubated in a 37° C. incubatorsupplied with 5% carbon dioxide. Cytopathic effects were observed in theinfected cells after 2 to 4 days of incubation. The infected cells werepassaged into new FRhK-4 cells and cytopathic effects were observedwithin 1 day after the inoculation. The infected cells were tested by animmunofluorescent assay for influenza A., influenza B, respiratorysyncytial virus, parainfluenza types 1, 2 and 3, adenovirus and humanmetapneumovirus (hMPV) and negative results were obtained for all cases.The infected cells were also tested by RT-PCR for influenza A and humanmetapneumovirus with negative results.

6.4 Virus Morphology

The infected cells prepared as described above were harvested, pelletedby centrifugation and the cell pellets were processed for thin-sectiontransmitted electron microscopic visualization. Viral particles wereidentified in the cells infected with both clinical specimens, but notin control cells which were not infected with the virus. Virionsisolated from the infected cells were about 70-100 nanometers (FIG. 2).Viral capsids were found predominantly within the vesicles of the golgiand endoplasmic reticulum and were not free in the cytoplasm. Virusparticles were also found at the cell membrane.

One virus isolate was ultracentrifuged and the cell pellet wasnegatively stained using phosphotugstic acid. Virus particlescharacteristic of Coronaviridae were thus visualized. Since the humanCoronaviruses hitherto recognized are not known to cause a similardisease, the present inventors postulated that the virus isolatesrepresent a novel virus that infects humans.

6.5 Antibody Response to the Isolated Virus

To further confirm that this novel virus is responsible for causing SARSin the infected patients, blood serum samples from the patients who weresuffering from SARS were obtained and a neutralization test wasperformed. Typically diluted serum (×50, ×200, ×800 and ×1600) wasincubated with acetone-fixed FRhK-4 cells infected with hSARS at 37° C.for 45 minutes. The incubated cells were then washed withphosphate-buffered saline and stained with anti-human IgG-FITCconjugated antibody. The cells were then washed and examined under afluorescent microscope. In these experiments, positive signals werefound in 8 patients who had SARS (FIG. 3), indicating that thesepatients had an IgG antibody response to this novel human respiratoryvirus of Coronaviridae. By contrast, no signal was detected in 4negative-control paired sera. The serum titers of anti-hSARS antibodiesof the tested patients are shown in Table 1.

TABLE 1 Name Date Lab No. Anti-SARS Patient A 25 Feb. 2003 S2728 <50 6Mar. 2003 S2728 1600 Patient B 26 Feb. 2003 S2441 50 3 Mar. 2003 S2441200 Patient C 4 Mar. 2003 S3279 200 14 Mar. 2003 S3279 1600 Patient D 6Mar. 2003 M41045 <50 11 Mar. 2003 MB943703 800 Patient E 4 Mar. 2003M38953 <50 18 Mar. 2003 KWH03/3601 800 Control F 13 Feb. 2003 M27124 <501 Mar. 2003 MB942968 <50 Patient G 3 Mar. 2003 M38685 <50 7 Mar. 2003KWH03/2900 Equivocal Blinded samples: 1a * Acute <50 1b Convalescent1600 2a * Acute 50 2b Convalescent >1600 3a * Acute 50 3bConvalescent >1600 4a * Acute <50 4b Convalescent <50 5a * Acute <50 5bConvaelscent <50 6a * Acute <50 6b Convalescent <50 NB: * patients withSARSNB: * patients with SARS

These results indicated that this novel member of Coronaviridae is a keypathogen in SARS.

6.6 Sequences of the hSARS Virus

Total RNA from infected or uninfected FrHK-4 cells was harvested twodays post-infection. One-hundred ng of purified RNA was reversetranscribed using Superscript® II reverse transcriptase (Invitrogen) ina 20 μl reaction mixture containing 10 pg of a degenerated primer(5′-GCCGGAGCTCTGCAGAATTCNNNNNN-3′: SEQ ID NO:5; N=A, T, G or C) asrecommended by the manufacturer. Reverse transcribed products were thenpurified by a QIAquick® PCR purification kit as instructed by themanufacturer and eluted in 30 μl of 10 mM Tris-HCl, pH 8.0. Three μl ofpurified cDNA products were add in a 25 μl reaction mixture containing2.5 μl of 10×PCR buffer, 4 μl of 25 mM MgCl₂, 0.5 μl of 10 mM dNTP, 0.25μl of AmpliTaq Gold® DNA polymerase (Applied Biosystems), 2.5 μCi of[α-³²P]CTP (Amersham), 2 μl of 10 μM primer(5′-GCCGGAGCTCTGCAGAATT-C-3′: SEQ ID NO:6). Reactions were thermalcycled through the following profile: 94° C. for 8 min followed by 2cycles of 94° C. for 1 min, 40° C. for 1 min, 72° C. for 2 min. Thistemperature profile was followed by 35 cycles of 94° C. for 1 min, 60°C. for 1 min, 72° C. for 1 min. 6 μl of the PCR products were analyzedin a 5% denaturing polyacrylamide gel electrophoresis. Gel was exposedto X-ray film and the film was developed after an over-night exposure.Unique PCR products which were only identified in infected cell sampleswere isolated from the gel and eluted in a 50 μl of 1×TE buffer. ElutedPCR products were then re-amplified in 25 μl of reaction mixturecontaining 2.5 μl of 10×PCR buffer, 4 μl of 25 mM MgCl₂, 0.5 μl ru 10 mMdNTP, 0.25 μl of AmpliTaq Gold® DNA polymerase (Applied Biosystems), 1μl of 10 μM primer (5′-GCCGGAGCTCTGCAGAATTC-3′:SEQ ID NO:6). Reactionmixtures were thermal cycled through the following profile: 94° C. for 8min followed by 35 cycles of 94° C. for 1 min, 60° C. for 1 min, 72° C.for 1 min. PCR products were cloned using a TOPO TA Cloning® kit(Invitrogen) and ligated plasmids were transformed into TOP10 E. colicompetent cells (Invitrogen). PCR inserts were sequenced by a BigDyecycle sequencing kit as recommended by the manufacturer (AppliedBiosystems) and sequencing products were analyzed by an automaticsequencer (Applied Biosystems, model number 3770). The obtained sequence(SEQ ID NO:1) is shown in FIG. 1. The deducted amino acid sequence (SEQID NO:2) from the obtained DNA sequence showed 57% homology to thepolymerase protein of identified coronaviruses.

Similarly, two other partial sequences (SEQ ID NOS:11 and 13) anddeduced amino acid sequences (SEQ ID NOS:12 and 14, respectively) wereobtained from the hSARS virus and are shown in FIGS. 8 (SEQ ID NOS:11and 12) and 9 (SEQ ID NOS:13 and 14).

The entire genomic sequence of hSARS virus is shown in FIG. 10 (SEQ IDNO:15). The deduced amino acid sequences of SEQ ID NO:15 in all threeframes are shown in FIG. 11 (nucleotide sequences shown in SEQ IDNOS:16, 240 and 737; for amino acid sequences, see SEQ ID NO:17-239,241-736 and 738-1107). The deduced amino acid sequences of thecomplement of SEQ ID NO:15 in all three frames are shown in FIG. 12(nucleotide sequences shown in SEQ NOS:1108, 1590 and 1965; for aminoacid sequences, see SEQ ID NOS:1109-1589, 1591-1964 and 1966-2470).

6.7 Detection of hSARS Virus in Nasopharyngeal Aspirates

First, the nasopharyngeal aspirates (NPA) were examined by rapidimmunoflourescent antigen detection for influenza A and B, parainfluenzatypes 1, 2 and 3, respiratory syncytial virus and adenovirus (Chan K H,Maldeis N, Pope W, Yup A, Ozinskas A. Gill J, Seto W H, Shortridge K F,Peiris J S M. Evaluation of Directigen Fly A+B test for rapid diagnosisof influenza A and B virus infections. J Clin Microbiol. 2002; 40:1675-1680) and were cultured for conventional respiratory pathogens onMardin Darby Canine Kidney, LLC-Mk2, RDE, Hep-2 and MRC-5 cells(Wiedbrauk D L, Johnston S L G. Manual of clinical virology. RavenPress, New York. 1993). Subsequently, fetal rhesus kidney (FRhk-4) andA-549 cells were added to the panel of cell lines used. Reversetranscription polymerase chain reaction (RT-PCR) was performed directlyon the clinical specimen for influenza A (Fouchier R A, Bestebroer T M,Herfst S, Van Der Kemp L, Rimmelzwan G F, Osterhaus A D. Detection ofinfluenza A virus from different species by PCR amplification ofconserved sequences in the matrix gene. J Clin Microbiol. 2000; 38:4096-101) and human metapneumovirus (HMPV). The primers used for HMPVwere: for first round, 5′-AARGTSAATGCATCAGC-3′ (SEQ ID NO. 7) and5′-CAKATTYTGCTTATGCTTTC-3′ (SEQ ID NO:8); and nested primers:5′-ACACCTGTTACAATACCAGC-3′ (SEQ ID NO:9) and 5′-GACTTGAGTCCCAGCTCCA-3′(SEQ ID NO:10). The size of the nested PCR product was 201 bp. An ELISAfor mycoplasma was used to screen cell cultures (Roche Diagnostics GmbH,Roche, Indianapolis, USA).

RT-PCR Assay

Subsequent to culturing and genetic sequencing of the hSARS virus fromtwo patients (see Section 6.6, supra), an RT-PCR was developed to detectthe hSARS virus sequence from NPA samples. Total RNA from clinicalsamples was reverse transcribed using random hexamers and cDNA wasamplified using primers 5′-TACACACCTCAGC-GTTG-3′ (SEQ ID NO:3) and5′-CACGAACGTGACGAAT-3′ (SEQ ID NO:4), which are constructed based on theRNA-dependent RNA polymerase-encoding sequence (SEQ ID NO:1) of thehSARS virus in the presence of 2.5 mM MgCl₂ (94° C. for 8 min followedby 40 cycles of 94° C. for 1 min, 50° C. for 1 min, 72° C. for 1 min).

The summary of a typical RT-PCR protocol is as follows:

1. RNA extraction

RNA from 140 μl of NPA samples is extracted by QIAquick viral RNAextraction kit and is eluted in 50 μl of elution buffer.

2. Reverse transcription

RNA 11.5 μl

0.1M DTT 2 μl

5× buffer 4 μl

10 mM dNTP 1 μl

Superscript II, 200 U/μl (Invitrogen) 1 μl

Random hexamers, 0.3 μg/μl 0.5 μl

Reaction condition 42° C., 50 min

-   -   94° C., 3 min    -   4° C.

3. PCR

cDNA generated by random primers is amplified in a 50 ul reaction asfollows:

cDNA 2 μl

10 mM dNTP 0.5 μl

10× buffer 5 μl

25 mM MgCl₂ 5 μl

25 μM Forward primer 0.5 μl

25 μM Reverse primer 0.5 μl

AmpliTaq Gold® polymerase, 5 U/μl (Applied Biosystems) 0.25 μl

Water 36.25 μl

Thermal-cycle condition: 95° C., 10 min, followed by 40 cycles of 95°C., 1 min; 50° C. 1 min; 72° C., 1 min.

4. Primer sequences

Primers were designed based on the RNA-dependent RNA polymerase encodingsequence (SEQ ID NO:1) of the hSARS virus.

Forward primer: 5′ TACACACCTCAGCGTTG 3′ (SEQ ID NO:3)

Reverse primer: 5′ CACGAACGTGACGAAT 3′ (SEQ ID NO:4)

Product size: 182 bps

Real-Time Quantitative PCR Assay

Total RNA from 140 μl of nasopharyngeal aspirate (NPA) was extracted byQIAamp® virus RNA mini kit (Qiagen) as instructed by the manufacturer.Ten μl of eluted RNA samples were reverse transcribed by 200 U ofSuperscript® II reverse transcriptase (Invitrogen) in a 20 μl reactionmixture containing 0.15 μg of random hexamers, 10 mmol/L DTT, and 0.5mmol/L dNTP, as instructed. Complementary DNA was then amplified in aSYBR® Green I fluorescence reaction (Roche) mixtures. Briefly, 20 μlreaction mixtures containing 2 μl of cDNA, 3.5 mmol/L MgCl₂, 0.25 μmol/Lof forward primer (5′-TACACACCTCAGCGTTG-3′; SEQ ID NO:3) and 0.25 μmol/Lreverse primer (5′-CACGAACGTGACGAAT-3′; SEQ ID NO:4) were thermal-cycledby a Light-Cycler (Roche) with the PCR program, [95° C., 10 min followedby 50 cycles of 95° C., 10 min; 57° C., 5 sec; 72° C. 9 sec]. Plasmidscontaining the target sequence were used as positive controls.Fluorescence signals from these reactions were captured at the end ofextension step in each cycle (see FIG. 7A). To determine the specificityof the assay, PCR products (184 base pairs) were subjected to a meltingcurve analysis at the end of the assay (65° C. to 95° C., 0.1° C. persecond; see FIG. 7B).

Clinical Results

Clinical Findings:

All 50 patients with SARS were ethnic Chinese. They represented 5different epidemiologically linked clusters as well as additionalsporadic cases fitting the case definition. They were hospitalized at amean of 5 days after the onset of symptoms. The median age was 42 years(range of 23 to 74) and the female to male ratio was 1.3. Fourteen (28%)were health care workers and five (10%) had a history of visit to ahospital experiencing a major outbreak of SARS. Thirteen (26%) patientshad household contacts and 12 (24%) others had social contacts withpatients with SARS. Four (8%) had a history of recent travel to mainlandChina.

The major complaints from most patients were fever (90%) and shortnessof breath. Cough and myalgia were present in more than half the patients(Table 2). Upper respiratory tract symptoms such as rhinorrhea (24%) andsore throat (20%) were present in a minority of patients. Diarrhea (10%)and anorexia (10%) were also reported. At initial examination,auscultatory findings, such as crepitations and decreased air entry,were present in only 38% of patients. Dry cough was reported by 62% ofpatients. All patients had radiological evidence of consolidation, atthe time of admission, involving 1 zone (in 36), 2 zones (13) and 3zones (1).

TABLE 2 Clinical symptoms Number (percentage) Fever 50 (100%) Chill orrigors 37 (74%) Cough 31 (62%) Myalgia 27 (54%) Malaise 25 (50%) Runningnose 12 (24%) Sore throat 10 (20%) Shortness of breath 10 (20%) Anorexia10 (20%) Diarrhea  5 (10%) Headache 10 (20%) Dizziness  6 (12%) *Truncal maculopapular rash was noted in 1 patient.

In spite of the high fever, most patients (98%) had no evidence of aleukocytosis. Lymphopenia (68%), leucopenia (26%), thrombocytopenia(40%) and anemia (18%) were present in peripheral blood examination(Table 3). Parenchymal liver enzyme, alanine aminotransferase (ALT) andmuscle enzyme, creatinine kinase (CPK) were elevated in 34% and 26%respectively.

TABLE 3 Laboratory parameter Mean (range) Percentage of bnormal Normalrange Haemoglobin 12.9 (8.9-15.9) 11.5-16.5 g/dl Anaemia  9 (18%) Whitecell count 5.17 (1.1-11.4) 4-11 x 10⁹/L Leucopenia 13 (26%) Lymphocytecount 0.78 (0.3-1.5) 1.5-4.0 x 10⁹/L Significant lymphopenia 34 (68%)(<1.0 × 10⁹/L) Platelet count  174 (88-351) 150-400 x 10⁹/LThrombocytopenia 20 (40%) Alanine aminotransaminase   63 (11-350) 6-53U/L (ALT) Elevated ALT 17 (34%) Albumin   37 (26-50) 42-54 g/L Lowalbumin 34 (68%) Globulin   33 (21-42) 24-36 g/L Elevated globulin 10(20%) Creatinine kinase  244 (31-1379) 34-138 U/L Elevated creatininekinase 13 (26%)

Routine microbiological investigations for known viruses and bacteria byculture, antigen detection, and PCR were negative in most cases. Bloodculture was positive for Escherichia coli in a 74-year-old male patient,who was admitted to intensive care unit, and was attributed to hospitalacquired urinary tract infection. Klebsiella pneumoniae and Hemophilusinfluenzae were isolated from the sputum specimens of 2 other patientson admission.

Oral levofloxacin 500 mg q24 h was given in 9 patients and intravenous(1.2 g q8 h)/oral (375 mg tid) amoxicillin-clavulanate andintravenous/oral clarithromycin 500 mg q12 h were given in another 40patients. Four patients were given oral oseltamivir 75 mg bid. In onepatient, intravenous ceftriaxone 2 gm q24 h, oral azithromycin 500 mgq24 h, and oral amantadine 100 mg bid were given for empirical coverageof typical and atypical pneumonia.

Nineteen patients progressed to severe disease with oxygen desaturationand were required intensive care and ventilatory support. The meannumber of days of deterioration from the onset of symptoms was 8.3 days.Intravenous ribavirin 8 mg/kg q8 h and steroid was given in 49 patientsat a mean day of 6.7 after onset of symptoms.

The risk factors associated with severe complicated disease requiringintensive care and ventilatory support were older age, lymphopenia,impaired ALT, and delayed initiation of ribavirin and steroid (Table 4).All the complicated cases were treated with ribavirin and steroid afteradmission to the intensive care unit whereas all the uncomplicated caseswere started on ribavirin and steroid in the general ward. As expected,31 uncomplicated cases recovered or improved whereas 8 complicated casesdeteriorated with one death at the time of writing. All 50 patients weremonitored for a mean of 12 days at the time of writing.

TABLE 4 Complicated Uncomplicated case case (n = 19) (n = 31) P valueMean (SD) age (range) 49.5 ± 12.7 39.0 ± 10.7 P < 0.01 Male/Female ratio8/11 14/17 N.S. Underlying illness 5^(†) 1^(‡) P < 0.05 Mode of contactTravel to China 1 3 N.S. Health care worker 5 9 N.S. Hospital visit 1 4N.S. Household contact 8 5 P < 0.05 Social contact 4 10 N.S. Mean (SD)duration of 5.2 ± 2.0 4.7 ± 2.5 N.S. symptoms to admission (days) Mean(SD) admission 38.8 ± 0.9 38.7 ± 0.8 N.S. temperature (° C.) Mean (SD)initial total 5.1 ± 2.4 5.2 ± 1.8 N.S. peripheral WBC count(× 10⁹/L)Mean (SD) initial lymphocyte 0.66 ± 0.3 0.85 ± 0.3 P < 0.05 count (×10⁹/L) Presence of thrombocytopenia 8 12 N.S. (<150 × 10⁹/L) Impairedliver function test 11 6 P < 0.01 CXR changes (number of 1.4 1.2 N.S.zone affected) Mean (SD) day of 8.3 ± 2.6 Not applicable deteriorationfrom the onset of symptoms § Mean (SD) day of initiation of 7.7 ± 2.95.7 ± 2.6 P < 0.05 Ribavirin & steroid from the onset of symptomsInitiation of ribavirin & 12 0 P < 0.001 steroid after deteriorationResponse to ribavirin & 11 28 P < 0.05 steroid Outcome Improved orrecovered 10 31 P < 0.01 Not improving ∥ 8 0 P < 0.01 * Multi-variantanalysis is not performed due to low number of cases; ^(†)2 patients haddiabetic mellitus, 1 had hypertrophic ostructive cardiomyopathy, 1 hadchronic active hepatitis B, and 1 had brain tumour; ^(‡)1 patient hadessential hypertension; § desaturation requiring intensive care support;∥ 1 died.

Two virus isolates, subsequently identified as a member of Coronaviridae(see below), were isolated from two patients. One was from an open lungbiopsy tissue of a 53-year-old Hong Kong Chinese resident and the otherfrom a nasopharyngeal aspirate of a 42 year-old female with goodprevious health. The 53-year old male had a history of 10-hour householdcontact with a Chinese visitor who came from Guangzhou and later diedfrom SARS. Two days after this exposure, he presented with fever,malaise, myalgia, and headache. Crepitations were present over the rightlower zone and there was a corresponding alveolar shadow on the chestradiograph. Hematological investigation revealed lymphopenia of0.7×109/L with normal total white cell and platelet counts. Both ALT (41U/L) and CPK (405 U/L) were impaired. Despite a combination of oralazithromycin, amantadine, and intravenous ceftriaxone, there wasincreasing bilateral pulmonary infiltrates and progressive oxygendesaturation. Therefore, an open lung biopsy was performed 9 days afteradmission. Histopathological examination showed a mild interstitialinflammation with scattered alveolar pneumocytes showing cytomegaly,granular amphophilic cytoplasm and enlarged nuclei with prominentnucleoli. No cells showed inclusions typical of herpesvirus oradenovirus infection. The patient required ventilation and intensivecare after the operative procedure. Empirical intravenous ribavirin andhydrocortisone were given. He succumbed 20 days after admission. Inretrospect, coronavirus-like RNA was detected in his nasopharyngealaspirate, lung biopsy and post-mortem lung. He had a significant rise intiter of antibodies against his own hSARS isolate from 1/200 to 1/1600.

The second patient from whom a hSARS virus was isolated, was a42-year-old female with good past health. She had a history of travel toGuangzhou in mainland China for 2 days. She presented with fever anddiarrhea 5 days after her return to Hong Kong. Physical examinationshowed crepitation over the right lower zone which had a correspondingalveolar shadow on the chest radiograph. Investigation revealedleucopenia (2.7×109/L), lymphopenia (0.6×109/L), and thrombocytopenia(104×109/L). Despite the empirical antimicrobial coverage withamoxicillin-clavulanate, clarithromycin, and oseltamivir, shedeteriorated 5 days after admission and required mechanical ventilationand intensive care for 5 days. She gradually improved without receivingtreatment with ribavirin or steroid. Her nasopharyngeal aspirate waspositive for the virus in the RT-PCR and she was seroconverted fromantibody titre <1/50 to 1/1600 against the hSARS isolate.

Virological Findings:

Viruses were isolated on FRhk-4 cells from the lung biopsy andnasopharyngeal aspirate respectively, of two patients described above.The initial cytopathic effect appeared between 2 and 4 days afterinoculation, but on subsequent passage, cytopathic effect appeared in 24hours. Both virus isolates did not react with the routine panel ofreagents used to identify virus isolates including those for influenzaA, B parainfluenza types 1,2,3, adenovirus and respiratory syncytialvirus (DAKO, Glostrup, Denmark). They also failed to react in RT-PCRassays for influenza A and HMPV or in PCR assays for mycoplasma. Thevirus was ether sensitive, indicating that it was an enveloped virus.Electron microscopy of negatively stained (2% potassiumphospho-tungstate, pH 7.0) cell culture extracts obtained byultracentrifugation showed the presence of pleomorphic enveloped viralparticles, of about 80-90 nm (ranging 70-130 nm) in diameter, whosesurface morphology appeared comparable to members of Coronaviridae (FIG.5A). Thin section electron microscopy of infected cells revealed virusparticles of 55-90 nm diameter within the smooth-walled vesicles in thecytoplasm (FIGS. 5A and 5B). Virus particles were also seen at the cellsurface. The overall findings were compatible with infections in thecells caused by viruses of Coronaviridae.

A thin section electron micrograph of the lung biopsy of the 53 year oldmale contained 60-90-nm viral particles in the cytoplasm of desquamatedcells. These viral particles were similar in size and morphology tothose observed in the cell-cultured virus isolate from both patients(FIG. 4).

The RT-PCR products generated in a random primer RT-PCR assay wereanalyzed and unique bands found in the virus infected specimen wascloned and sequenced. Of 30 clones examined, a clone containing 646 basepairs (SEQ ID NO:1) of unknown origin was identified. Sequence analysisof this DNA fragment suggested this sequence had a weak homology toviruses of the family of Coronaviridae (data not shown). Deducted aminoacid sequence (215 amino acids: SEQ ID NO:2) from this unknown sequence,however, had the highest homology (57%) to the RNA polymerase of bovinecoronavirus and murine hepatitis virus, confirming that this virusbelongs to the family of Coronaviridae. Phylogenetic analysis of theprotein sequences showed that this virus, though most closely related tothe group II coronaviruses, was a distinct virus (FIGS. 5A and 5B).

Based on the 646 by sequence of the isolate, specific primers fordetecting the new virus was designed for RT-PCR detection of this hSARSvirus genome in clinical specimens. Of the 44 nasopharyngeal specimensavailable from the 50 SARS patients, 22 had evidence of hSARS RNA. ViralRNA was detectable in 10 of 18 fecal samples tested. The specificity ofthe RT-PCR reaction was confirmed by sequencing selected positive RT-PCRamplified products. None of 40 nasophararyngeal and fecal specimens frompatients with unrelated diseases were reactive in the RT-PCR assay.

To determine the dynamic range of real-time quantitative PCR, serialdilutions of plasmid DNA containing the target sequence were made andsubjected to the real-time quantitative PCR assay. As shown in FIG. 7A,the assay was able to detect as little as 10 copies of the targetsequence. By contrast, no signal was observed in the water control (FIG.7A). Positive signals were observed in 23 out of 29 serologicallyconfirmed SARS patients. In all of these positive cases, a unique PCRproduct (T_(m)=82° C.) corresponding to the signal from the positivecontrol was observed (FIG. 7B, and data not shown). These resultsindicated this assay is highly specific to the target. The copy numbersof the target sequence in these reactions range from 4539 to less than10. Thus, as high as 6.48×10⁵ copies of this viral sequence could befound in 1 ml of NPA sample. In 5 of the above positive cases, it waspossible to collect NPA samples before seroconvertion. Viral RNA wasdetected in 3 of these samples, indicating that this assay can detectthe virus even at the early onset of infection.

To further validate the specificity of this assay, NPA samples fromhealthy individuals (n=11) and patients suffered from adenovirus (n=11),respiratory syncytial virus (n=11), human metapneumovirus (n=11),influenza A virus (n=13) or influenza B virus (n=1) infection wererecruited as negative controls. All of these samples, except one, werenegative in the assay. The false positive case was negative in asubsequence test. Taken together, including the initial false positivecase, the real-time quantitative PCR assay has sensitivity of 79% andspecificity of 98%.

Epidemiological data suggest that droplet transmission is one of themajor route of transmission of this virus. The detection of live virusand the detection of high copies of viral sequence from NPA samples inthe current study clearly support that cough and sneeze droplets fromSARS patients might be the major source of this infectious agent.Interestingly, 2 out of 4 available stool samples form the SARA patientsin this study were positive in the assay (data not shown). The detectionof the virus in feces suggests that there might be other routes oftransmission. It is relevant to note that a number of animalcoronaviruses are spread via the fecal-oral route (McIntosh K., 1974,Coronaviruses: a comparative review. Current Top Microbiol Immunol. 63:85-112). However, further studies are required to test whether the virusin feces is infectious or not.

Currently, apart form this hSARS virus, there are two known serogroupsof human coronaviruses (229E and OC43) (Hruskova J. et al., 1990,Antibodies to human coronaviruses 229E and OC43 in the population ofC.R., Acta Virol. 34:346-52). The primer set used in the present assaydoes not have homology to the strain 229E. Due to the lack of availablecorresponding OC43 sequence in the Genebank, it is not known whetherthese primers would cross-react with this strain. However, sequenceanalyses of available sequences in other regions of OC43 polymerase geneindicate that the novel human virus associated with SARS is geneticallydistinct from OC43. Furthermore, the primers used in this study do nothave homology to any of sequences from known coronaviruses. Thus, it isvery unlikely that these primers would cross-react with the strain OC43.

Apart from the novel pathogen, metapneumovirus was reported to beidentified in some of SARS patients (Center for Disease Control andPrevention, 2003, Morbidity and Mortality Weekly Report 52: 269-272). Noevidence of metapneumovirus infection was detected in any of thepatients in this study (data not shown), suggesting that the novel hSARSvirus of the invention is the key player in the pathogenesis of SARS.

Immunofluorescent Antibody Detection:

Thirty-five of the 50 most recent serum samples from patients with SARShad evidence of antibodies to the hSARS (see FIG. 3). Of 27 patientsfrom whom paired acute and convalescent sera were available, all wereseroconverted or had >4 fold increase in antibody titer to the virus.Five other pairs of sera from additional SARS patients from clustersoutside this study group were also tested to provide a wider sampling ofSARS patients in the community and all of them were seroconverted. Noneof 80 sera from patients with respiratory or other diseases as well asnone of 200 normal blood donors had detectable antibody.

When either seropositivity to HP-CV in a single serum or viral RNAdetection in the NPA or stool are considered evidence of infection withthe hSARS, 45 of the 50 patients had evidence of infection. Of the 5patients without any virological evidence of Coronaviridae viralinfection, only one of these patients had their sera tested >14 daysafter onset of clinical disease.

DISCUSSION

The outbreak of SARS is unusual in a number of aspects, in particular,in the appearance of clusters of patients with pneumonia in health careworkers and family contacts. In this series of patients with SARS,investigations for conventional pathogens of atypical pneumonia provednegative. However, a virus that belongs to the family Coronaviridae wasisolated from the lung biopsy and nasopharyngeal aspirate obtained fromtwo SARS patients, respectively. Phylogenetically, the virus was notclosely related to any known human or animal coronavirus or torovirus.The present analysis is based on a 646 by fragment (SEQ ID NO:1) of thepolymerase gene and the entire genome of the isolated hSARS virus, whichindicates that the virus relates to antigenic group 2 of thecoronaviruses along with murine hepatitis virus and bovine coronavirus.However, viruses of the Coronaviridae can undergo heterologousrecombination within the virus family and genetic analysis of otherparts of the genome needs to be carried out before the nature of thisnew virus is more conclusively defined (Holmes K V. Coronaviruses. EdsKnipe D M, Howley P M Fields Virology, 4th Edition, Lippincott Williams& Wilkins, Philadelphia, 1187-1203). The biological, genetic andclinical data, taken together, indicate that the new virus is not one ofthe two known human coronaviruses.

The majority (90%) of patients with clinically defined SARS had eitherserological or RT-PCR evidence of infection by this virus. In contrast,neither antibody nor viral RNA was detectable in healthy controls. All27 patients from whom acute and convalescent sera were availabledemonstrated rising antibody titers to hSARS virus, strengthening thecontention that a recent infection with this virus is a necessary factorin the evolution of SARS. In addition, all five pairs of acute andconvalescent sera tested from patients from other hospitals in Hong Kongalso showed seroconversion to the virus. The five patients who has notshown serological or virological evidence of hSARS virus infection, needto have later convalescent sera tested to define if they are alsoseroconverted. However, the concordance of the hSARS virus with theclinical definition of SARS appears remarkable, given that clinical casedefinitions are never perfect.

No evidence of HMPV infection, either by RT-PCR or rising antibody titeragainst HMPV, was detected in any of these patients. No other pathogenwas consistently detected in our group of patients with SARS. It istherefore highly likely that that this hSARS virus is either the causeof SARS or a necessary pre-requisite for disease progression. Whether ornot other microbial or other co-factors play a role in progression ofthe disease remains to be investigated.

The family Coronaviridae includes the genus Coronavirus and Torovirus.They are enveloped RNA viruses which cause disease in humans andanimals. The previously known human coronaviruses, types 229E and OC43are the major causes of the common cold (Holmes K V. Coronaviruses. EdsKnipe D M, Howley P M Fields Virology, 4th Edition, Lippincott Williams& Wilkins, Philadelphia, 1187-1203). But, while they can occasionallycause pneumonia in older adults, neonates or immunocompromised patient(El-Sahly H M, Atmar R L, Glezen W P, Greenberg S B. Spectrum ofclinical illness in hospitalized patients with “common cold” virusinfections. Clin Infect Dis. 2000; 31: 96-100; and Foltz E J, Elkordy MA. Coronavirus pneumonia following autologous bone marrowtransplantation for breast cancer. Chest 1999; 115: 901-905),Coronaviruses have been reported to be an important cause of pneumoniain military recruits, accounting for up to 30% of cases in some studies(Wenzel R P, Hendley J O, Davies J A, Gwaltney J M, Coronavirusinfections in military recruits: Three-year study with coronavirusstrains OC43 and 229E. Am Rev Respir Dis. 1974; 109: 621-624). Humancoronaviruses can infect neurons and viral RNA has been detected in thebrain of patients with multiple sclerosis (Talbot P J, Cote G, Arbour N.Human coronavirus OC43 and 229E persistence in neural cell cultures andhuman brains. Adv Exp Med. Biol.-in press). On the other hand, a numberof animal coronaviruses (eg. Porcine Transmissible GastroenteritisVirus, Murine Hepatitis Virus, Avian Infectious Bronchititis Virus)cause respiratory, gastrointestinal, neurological or hepatic disease intheir respective hosts (McIntosh K. Coronaviruses: a comparative review.Current Top Microbiol Immunol. 1974; 63: 85-112).

We describe for the first time the clinical presentation andcomplications of SARS. Less than 25% of patients with coronaviralpneumonia had upper respiratory tract symptoms. As expected in atypicalpneumonia, both respiratory symptoms and positive auscultatory findingswere very disproportional to the chest radiographic findings.Gastrointestinal symptoms were present in 10%. It is relevant that thevirus RNA is detected in faeces of some patients and that coronaviruseshave been associated with diarrhoea in animals and humans (Caul E O,Egglestone S I. Further studies on human enteric coronaviruses ArchVirol. 1977; 54: 107-17). The high incidence of deranged liver functiontest, leucopenia, significant lymphopenia, thrombocytopenia andsubsequent evolution into adult respiratory distress syndrome suggests asevere systemic inflammatory damage induced by this hSARS virus. Thusimmuno-modulation by steroid may be important to complement theantiviral therapy by ribavirin. In this regard, it is pertinent thatsevere human disease associated with the avian influenza subtype H5N1,another virus that recently crossed from animals to humans, has alsobeen postulated to have an immuno-pathological component (Cheung C Y,Poon L L M, Lau A S Y et al. Induction of proinflammatory cytokines inhuman macrophages by influenza A (H5N1) viruses: a mechanism for theunusual severity of human disease. Lancet 2002; 360: 1831-1837). Incommon with H5N1 disease, patients with severe SARS are adults, aresignificantly more lymphopenic and have parameters of organ dysfunctionbeyond the respiratory tract (Table 4) (Yuen K Y, Chan P K S, Peiris J SM, et al. Clinical features and rapid viral diagnosis of human diseaseassociated with avian influenza A H5N1 virus. Lancet 1998; 351:467-471). It is important to note that a window of opportunity of around8 days exists from the onset of symptoms to respiratory failure. Severecomplicated cases are strongly associated with both underlying diseaseand delayed use of ribavirin and steroid therapy. Following our clinicalexperience in the initial cases, this combination therapy was startedvery early in subsequent cases which were largely uncomplicated cases atthe time of admission. The overall mortality at the time of writing isonly 2% with this treatment regimen. There were still 8 out of 19complicated cases who had not shown significant response. It is notpossible to a detail analysis of the therapeutic response to thiscombination regimen due to the heterogeneous dosing and time ofinitiation of therapy.

Other factors associated with severe disease is acquisition of thedisease through household contact which may be attributed to a higherdose or duration of viral exposure and the presence of underlyingdiseases.

The clinical description reported here pertains largely to the moresevere cases admitted to hospital. We presently have no data on the fullclinical spectrum of the emerging Coronaviridae infection in thecommunity or in an out-patient-setting. The availability of diagnostictests as described here will help address these questions. In addition,it will allow questions pertaining to the period of virus shedding (andcommunicability) during convalescence, the presence of virus in otherbody fluids and excreta and the presence of virus shedding during theincubation period, to be addressed.

The epidemiological data at present appears to indicate that the virusis spread by droplets or by direct and indirect contact althoughairborne spread cannot be ruled out in some instances. The finding ofinfectious virus in the respiratory tract supports this contention.Preliminary evidence also suggests that the virus may be shed in thefeces. However, it is important to note that detection of viral RNA doesnot prove that the virus is viable or transmissible. If viable virus isdetectable in the feces, this would be a potentially additional route oftransmission that needs to be considered. It is relevant to note that anumber of animal coronaviruses are spread via the fecal-oral route(McIntosh K. Coronaviruses: a comparative review. Current Top MicrobiolImmunol. 1974; 63: 85-112).

In conclusion, this report provides evidence that a virus in theCoronaviridae family is the etiological agent of SARS.

7. DEPOSIT

A sample of isolated hSARS virus was deposited with China Center forType Culture Collection (CCTCC) at Wuhan University, Wuhan 430072 inChina on Apr. 2, 2003 in accordance with the Budapest Treaty on theDeposit of Microorganisms, and accorded accession No. CCTCC-V200303,which is incorporated herein by reference in its entirety.

8. MARKET POTENTIAL

The hSARS virus can now be grown on a large scale, which allows thedevelopment of various diagnostic tests as described hereinabove as wellas the development of vaccines and antiviral agents that are effectivein preventing, ameliorating or treating SARS. Given the severity of thedisease and its rapid global spread, it is highly likely thatsignificant demands for diagnostic tests, therapies and vaccines tobattle against the disease, will arise on a global scale. In addition,this virus contains genetic information which is extremely important andvaluable for clinical and scientific research applications.

9. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain manyequivalents to the specific embodiments of the invention describedherein using no more than routine experimentation. Such equivalents areintended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

1. A formulation comprising a cell culture extract of an isolated humanSevere Acute Respiratory Syndrome (hSARS) virus having China Center forType Culture Collection Deposit Accession No. CCTCC-V200303.
 2. A kitcomprising a container containing the formulation of claim 1.